Method for modifying plant morphology, biochemistry and physiology

ABSTRACT

The present invention relates to methods for stimulating root growth and/or enhancing the formation of lateral or adventitious roots and/or altering root geotropism comprising expression of a plant cytokinin oxidase or comprising expression of another protein that reduces the level of active cytokinins in plants or plant parts. The invention also relates to novel plant cytokinin oxidase proteins, nucleic acid sequences encoding cytokinin oxidase proteins as well as to vectors, host cells, transgenic cells and plants comprising said sequences. The invention also relates to the use of said sequences for improving root-related characteristics including increasing yield and/or enhancing early vigor and/or modifying root/shoot ratio and/or improving resistance to lodging and/or increasing drought tolerance and/or promoting in vitro propagation of explants and/or modifying cell fate and/or plant development and/or plant morphology and/or plant biochemistry and/or plant physiology. The invention also relates to the use of said sequences in the above-mentioned methods. The invention also relates to methods for identifying and obtaining proteins and compounds interacting with cytokinin oxidase proteins. The invention also relates to the use of said compounds as a plant growth regulator or herbicide.

FIELD OF THE INVENTION

[0001] The present invention generally relates to a method for modifyingplant morphological, biochemical and physiological properties orcharacteristics, such as one or more developmental processes and/orenvironmental adaptive processes, including but not limited to themodification of initiation or stimulation or enhancement of root growth,and/or adventitious root formation, and/or lateral root formation,and/or root geotropism, and/or shoot growth, and/or apical dominance,and/or branching, and/or timing of senescence, and/or timing offlowering, and/or flower formation, and/or seed development, and/or seedyield, said method comprising expressing a cytokinin degradation controlprotein, in particular cytokinin oxidase, in the plant, operably underthe control of a regulatable promoter sequence such as a cell-specificpromoter, tissue-specific promoter, or organ-specific promoter sequence.Preferably, the characteristics modified by the present invention arecytokinin-mediated and/or auxin-mediated characteristics. The presentinvention extends to genetic constructs which are useful for performingthe inventive method and to transgenic plants produced therewith havingaltered morphological and/or biochemical and/or physiological propertiescompared to their otherwise isogenic counterparts.

BACKGROUND OF THE INVENTION

[0002] Roots are an important organ of higher plants. Their mainfunctions are anchoring of the plant in the soil and uptake of water andnutrients (N-nutrition, minerals, etc.). Thus, root growth has a director indirect influence on growth and yield of aerial organs, particularlyunder conditions of nutrient limitation. Roots are also relevant for theproduction of secondary plant products, such as defense compounds andplant hormones.

[0003] Roots are also storage organs in a number of important staplecrops. Sugar beet is the most important plant for sugar production inEurope (260 Mill t/year; 38% of world production). Manioc (cassaya),yams and sweet potato (batate) are important starch producers (app. 150Mill t/year each). Their content in starch can be twice as high as thatof potato. Roots are also the relevant organ for consumption in a numberof vegetables (e.g. carrots, radish), herbs (e.g. ginger, kukuma) andmedicinal plants (e.g. ginseng). In addition, some of the secondaryplant products found in roots are of economic importance for thechemical and pharmaceutical industry. An example is yams, which containbasic molecules for the synthesis of steroid hormones. Another exampleis shikonin, which is produced by the roots of Lithospermumerythrorhizon in hairy root cultures. Shikonin is used for itsanti-inflammatory, anti-tumour and wound-healing properties.

[0004] Moreover, improved root growth of crop plants will also enhancecompetitiveness with weedy plants and will improve growth in arid areas,by increasing water accessibility and uptake.

[0005] Improved root growth is also relevant for ecological purposes,such as bioremediation and prevention/arrest of soil erosion.

[0006] Root architecture is an area that has remained largely unexploredthrough classical breeding, because of difficulties with assessing thistrait in the field. Thus, biotechnology could have significant impact onthe improvement of this trait, because it does not rely on large-scalescreenings in the field. Rather, biotechnological approaches require abasic understanding of the molecular components that determine aspecific characteristic of the plant. Today, this knowledge is onlyfragmentary, and as a consequence, biotechnology was so far unable torealize a break-through in this area.

[0007] A well-established regulator of root growth is auxin. Applicationof indole-3-acetic acid (IAA) to growing plants stimulates lateral rootdevelopment and lateral root elongation (Torrey, Am J Bot 37: 257-264,1950; Blakely et al., Bot Gaz 143: 341-352, 1982; Muday and Haworth,Plant Physiol Biochem 32: 193-203, 1994). Roots exposed to a range ofconcentrations of IAA initiated increasing numbers of lateral roots(Kerk et al., Plant Physiol, 122: 925-932, 2000). Furthermore, whenroots that had produced laterals in response to a particularconcentration of exogenous auxin were subsequently exposed to a higherconcentration of IAA, numerous supernumerary lateral roots spacedbetween existing ones were formed (Kerk et al., Plant Physiol, 122:925-932, 2000). Conversely, growth of roots on agar containingauxin-transport inhibitors, including NPA, decreases the number oflateral roots (Muday and Haworth, Plant Physiol Biochem 32: 193-203,1994).

[0008] Arabidopsis mutants containing Increased levels of endogenous IAAhave been isolated (Boerjan et al., Plant Cell 7: 1405-141, 1995;Celenza et al., Gene Dev 9: 2131-2142, 1995; King et al., Plant Cell 7:2023-2037, 1995; Lehman et al., Cell 85: 183-194, 1996). They are nowknown to be alleles of a single locus located on chromosome 2. Thesemutant seedlings have excess adventitious and lateral roots, which is inaccordance with the above-described effects of external auxinapplication.

[0009] The stimulatory effect of auxins on adventitious and lateral rootformation suggests that overproduction of auxins in transgenic plants isa valid strategy for increasing root growth. Yet, it is alsoquestionable whether this would yield a commercial product with improvedcharacteristics. Apart from its stimulatory effect on adventitious andlateral root formation, auxin overproduction triggers other effects,such as reduction in leaf number, abnormal leaf morphology (narrow,curled leaves), aborted inflorescences, increased apical dominance,adventitios root formation on the stem, most of which are undesirablefrom an agronomic perspective (Klee et al., Genes Devel 1: 8696, 1987;Kares et al., Plant Mol Biol 15: 225-236, 1990). Therefore, the majorproblem with approaches that rely on increased auxin synthesis is aproblem of containment, namely to confine the effects of auxin to theroot. This problem of containment is not likely overcome by usingtissue-specific promoters: auxins are transported in the plant and theiraction is consequently not confined to the site of synthesis. Anotherissue is whether auxins will always enhance the total root biomass. Foragar-grown plants, it has been noticed that increasing concentrationsprogressively stimulated lateral root formation but concurrentlyinhibited the outgrowth of these roots (Kerk et al., Plant Physiol, 122:925932, 2000).

[0010] The above-mentioned problems related to containment of auxineffects and to maintenance of root outgrowth are solved by theembodiments in the patent claim.

SUMMARY OF THE INVENTION

[0011] The present invention relates to a genetic construct comprising agene encoding a protein with cytokinin oxidase activity from Arabidopsisthaliana. This gene is expressed under control of a regulated promoter.This promoter may be regulated by endogenous tissue-specific orenvironment-specific factors or, alternatively, it may be induced byapplication of specific chemicals.

[0012] The present invention also relates to a cell or plant containingthe genetic construct.

[0013] The present invention also relates to a method to modify rootarchitecture and biomass by expression of a cytokinin oxidase gene undercontrol of a promoter that is specific to the root or to certain tissuesor cell types of the root.

DETAILED DESCRIPTION OF THE INVENTION

[0014] To by-pass above-mentioned problems associated with increasingauxin biosynthesis, it was decided to follow an alternative approach. Wereasoned that down-regulation of biological antagonists of auxins couldevoke similar or even superior effects on root growth as compared toincreasing auxin levels. Hormone actions and interactions are extremelycomplex, but we hypothesized that cytokinins could function as auxinantagonists with respect to root growth. Hormone studies on plant tissuecultures have shown that the ratio of auxin versus cytokinin is moreimportant for organogenesis than the absolute levels of each of thesehormones, which indeed indicates that these hormones function asantagonists—at least in certain biological processes. Furthermore,lateral root formation is inhibited by exogenous application ofcytokinins. Interestingly, also root elongation is negatively affectedby cytokinin treatment, which suggests that cytokinins control both rootbranching and root outgrowth.

[0015] Together, current literature data indicate that increasingcytokinin levels negatively affects root growth, but the mechanismsunderlying this process are not understood. The sites of cytokininsynthesis in the plant are root tips and young tissues of the shoot.Endogenous concentrations of cytokinins are in the nM range. However, astheir quantification is difficult, rather large tissue amounts need tobe extracted and actual local concentrations are not known. Also thesubcellular compartmentation of cytokinins is not known. It is generallythought that the free base and ribosides are localized in the cytoplasmand nucleus, while glucosides are localized in the vacuole. There existalso different cytokinins with slightly different chemical structure. Asa consequence, it is not known whether the effects of exogenouscytokinins should be ascribed to a raise in total cytokininconcentration or rather to the competing out of other forms ofplant-borne cytokinins (which differ either in structure, cellular orsubcellular location) for receptors, translocators, transporters,modifying enzymes . . .

[0016] In order to test the hypothesis that cytokinin levels in the rootindeed exceed the level optimal for root growth, novel genes encodingcytokinin oxidases (which are cytokinin metabolizing enzymes) werecloned from Arabidopsis thaliana (designated AtCKX) and weresubsequently expressed under a strong constitutive promoter intransgenic tobacco and Arabidopsis. Transformants showing AtCKX mRNAexpression and increased cytokinin oxidase activity also manifestedenhanced formation and growth of roots.

[0017] Negative effects on shoot growth were also observed. The latteris in accordance with the constitutive expression of the cytokininoxidase gene in these plants, illustrating the importance of confinedexpression of the cytokinin oxidase gene for general plant growthproperties. Containment of cytokinin oxidase actMty can be achieved byusing cell-, tissue- or organ-specific promoters, since cytokinindegradation is a process limited to the tissues or cells that expressthe CKX protein, this in contrast to approaches relying on hormonesynthesis, as explained above.

[0018] The observed negative effects of cytokinin oxidase expression onshoot growth demonstrate that cytokinin oxidases are interesting targetsfor the design of or screening for growth-promoting chemicals. Suchchemicals should inhibit cytokinin oxidase activity, should preferablynot be transported to the root and should be rapidly degraded in soil,so that application of these chemicals will not inhibit root growth.Cytokinins also delay leaf senescence, which means that positive effectswill include both growth and maintenance of photosynthetic tissues. Inaddition, the observation that cytokinins delay senescence, enhancegreening (chlorophyll content) of leaves and reduce shoot apicaldominance shows that strategies based on suppressing CKX activity (suchas antisense, ribozyme, and cosuppression technology) in the aerialparts of the plant could result in delayed senescence, enhanced leafgreening and increased branching.

[0019] Similarly, the observed positive effects of cytokinin oxidaseexpression on root growth demonstrate that cytokinin oxidases areinteresting targets for the design of or screening for herbicides. Suchherbicides should inhibit cytokinin oxidase activity, should preferablynot be transported to the shoot, and should be soluble and relativelystable in a solvent that can be administered to the root through thesoil.

[0020] These effects of cytokinin oxidase overexpression on plantdevelopment and architecture were hitherto unknown and, as aconsequence, the presented invention and its embodiments could not beenvisaged.

[0021] The observed negative effects on shoot growth demonstrate thatmanipulation of cytokinin oxidases can also be used for obtainingdwarfing phenotypes. Dwarfing phenotypes are particularly useful incommercial crops such as cereals and fruit trees for example.

[0022] Preferable embodiments of the invention relate to the positiveeffect of cytokinin oxidase expression on plant growth and architecture,and in particular on root growth and architecture. The cytokinin oxidasegene family contains at least six members in Arabidopsis (see examplesbelow) and the present inventors have shown that there are quantitativedifferences in the effects achieved with some of these genes intransgenic plants. It is anticipated that functional homologs of thedescribed Arabidopsis cytokinin oxidases can be isolated from otherorganisms, given the evidence for the presence of cytokinin oxidaseactivity in many green plants (Hare and van Staden, Physiol Plant91:128-136, 1994; Jones and Schreiber, Plant Growth Reg 23:123-134,1997), as well as in other organisms (Armstrong, in Cytokinins:Chemistry, Activity and Function. Eds Mok and Mok, CRC Press, pp139-154,1994). Therefore, the sequence of the cytokinin oxidase, functional inthe invention, need not to be identical to those described herein. Thisinvention is particularly useful for cereal crops and monocot crops ingeneral and cytokinin oxidase genes from for example wheat or maize maybe used as well (Morris et al., 1999; Rinaldi and Comandini, 1999). Itis envisaged that other genes with cytokinin oxidase activity or withany other cytokinin metabolizing activity (see Za{haeck over (z)}ímalováet al., Biochemistry and Molecular Biology of Plant Hormones, Hooykaas,Hall and Libbenga (Eds.), Elsevier Science, pp141-160, 1997) can also beused for the purpose of this invention. Similarly, genes encodingproteins that would increase endogenous cytokinin metabolizing activitycan also be used for the purpose of this invention. In principle,similar phenotypes could also be obtained by interfering with genes thatfunction downstream of cytokinin such as receptors or proteins involvedin signal transduction pathways of cytokinin.

[0023] For the purpose of this invention, it should be understood thatthe term ‘root growth’ encompasses all aspects of growth of thedifferent parts that make up the root system at different stages of itsdevelopment, both in monocotyledonous and dicotyledonous plants. It isto be understood that enhanced growth of the root can result fromenhanced growth of one or more of its parts including the primary root,lateral roots, adventitious roots, etc. all of which fall within thescope of this invention.

[0024] According to a first embodiment, the present invention relates toa method for stimulating root growth and/or enhancing the formation oflateral and/or adventitious roots and/or altering root geotropismcomprising expression of a plant cytokinin oxidase or comprisingexpression of another protein that reduces the level of activecytokinins in plants or plant parts.

[0025] In the context of the present invention it should be understoodthat the term “expression” and/or ‘overexpression’ are usedinterchangeably and both relate to an “enhanced and/or ectopicexpression” of a plant cytokinin oxidase or any other protein thatreduces the level of active cytokinins in plants. It should be clearthat herewith an enhanced expression of the plant cytokinin oxidase aswell as “de novo” expression of plant cytokinin oxidases or of saidother proteins is meant. Alternatively, said other protein enhances thecytokinin metabolizing activity of a plant cytokinin oxidase.

[0026] It futher should be understood that in the context of the presentinvention the expression “lateral and/or adventitious roots” can mean“lateral and adventitious roots” but also “lateral or adventitiousroots”. The enhancement can exist in the formation of lateral roots orin the formation of adventitious roots as well as in the formation ofboth types of non-primary roots, but not necessarily.

[0027] According to a further embodiment, the present invention relatesto a method for stimulating root growth and/or enhancing the formationof lateral or adventitious roots and/or altering root geotropism and/orincreasing yield and/or enhancing early vigor and/or modifyingroot/shoot ratio and/or improving resistance to lodging and/orincreasing drought tolerance and/or promoting in vitro propagation ofexplants, comprising expression of a plant cytokinin oxidase orcomprising expression of another protein that reduces the level ofactive cytokinins in plants or plant parts.

[0028] According to a preferred embodiment, the present inventionrelates to a method for stimulating root growth resulting in an increaseof root mass by overexpression of a cytokinin oxidase, preferably acytokinin oxidase according to the invention, or another protein thatreduces the level of active cytokinins in plants or plant parts,preferably in roots.

[0029] Higher root biomass production due to overexpression of growthpromoting sequences has a direct effect on the yield and an indirecteffect of production of compounds produced by root cells or transgenicroot cells or cell cultures of said transgenic root cells. One exampleof an interesting compound produced in root cultures is shikonin, theyield of which can be advantageously enhanced by said methods.

[0030] According to a more specific embodiment, the present inventionrelates to a method for stimulating root growth or for enhancing theformation of lateral and adventitious roots or for altering rootgeotropism comprising expression of a nucleic acid encoding a plantcytokinin oxidase selected from the group consisting of:

[0031] (a) nucleic acids comprising a DNA sequence as given in any ofSEQ ID NOs 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33 or 34, or thecomplement thereof,

[0032] (b) nucleic acids comprising the RNA sequences corresponding toany of SEQ ID NOs 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33 or 34, orthe complement thereof,

[0033] (c) nucleic acids specifically hybridizing to any of SEQ ID NOs27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33 or 34, or to the complementthereof,

[0034] (d) nucleic acids encoding a protein comprising the amino acidsequence as given in any of SEQ ID NOs 2, 4, 6, 8, 10, 12, 32 or 35, orthe complement thereof,

[0035] (e) nucleic acids as defined in any of (a) to (d) characterizedin that said nucleic acid is DNA, genomic DNA, cDNA, synthetic DNA orRNA wherein T is replaced by U,

[0036] (f) nucleic acids which are degenerated to a nucleic acid asgiven in any of SEQ ID NOs 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33or 34, or which are degenerated to a nucleic acid as defined in any of(a) to (e) as a result of the genetic code,

[0037] (g) nucleic acids which are diverging from a nucleic acidencoding a protein as given in any of SEQ ID NOs 2, 4, 6, 8, 10, 12 or35 or which are diverging from a nucleic acid as defined in any of (a)to (e), due to the differences in codon usage between the organisms,

[0038] (h) nucleic acids encoding a protein as given in SEQ ID NOs 2, 4,6, 8, 10, 12 or 35 or nucleic acids as defined in (a) to (e) which arediverging due to the differences between alleles,

[0039] (i) nucleic acids encoding a protein as given in any of SEQ IDNOs 2, 4, 6, 8, 10, 12 or 35,

[0040] (j) functional fragments of nucleic acids as defined in any of(a) to (i) having the biological activity of a cytokinin oxidase, and

[0041] (k) nucleic acids encoding a plant cytokinin oxidase,

[0042] or comprising expression, preferably in roots, of a nucleic acidencoding a protein that reduces the level of active cytokinins in plantsor plant parts.

[0043] In the present invention, nucleic acids encoding novelArabidopsis thaliana cytokinine oxidases have been isolated and for thefirst time, the present inventors suprisingly could show that theexpression of cytokinin oxidases in transgenic plants or in transgenicplant parts resulted in the above-mentioned root-related features.Preferably, the expression of the cytokinine oxidase(s) should takeplace in roots, preferably under the control of a root-specificpromoter. One example of such a root-specific promoter is provided inSEQ ID NO 36.

[0044] It should be clear that, although the invention is supported inthe examples section by several new AtCKX genes and proteins, theinventive concept also relates to the use of other cytokinin oxidasesisolated from and expressed in other plants, preferably in the roots ofsaid other plants to obtain similar effects in plants as desribed in theexamples section.

[0045] Therefore, the present invention more generally relates to theuse of a nucleic acid encoding a plant cytokinin oxidase or encoding aprotein that reduces the level of active cytokinins in plants or plantparts for stimulating root growth or for enhancing the formation oflateral or adventitious roots or for altering root geotropism. Preferredcytokinin oxidases to be used are encoded by the nucleic acids encodingthe cytokinin oxidases as defined above and are encoded by the novelnucleic acids of the invention as defined hereunder.

[0046] The invention relates to an isolated nucleic acid encoding anovel plant protein having cytokinin oxidase activity selected from thegroup consisting of:

[0047] (a) a nucleic acid comprising a DNA sequence as given in any ofSEQ ID NOs 29, 3, 5, 9, 26, 27, 31, 33 or 34, or the complement thereof,

[0048] (b) a nucleic acid comprising the RNA sequences corresponding toany of. SEQ ID NOs 29, 3, 5, 9, 26, 27, 31, 33 or 34, or the complementthereof,

[0049] (c) a nucleic acid specifically hybridizing to a nucleic acid asgiven in any of SEQ ID NOs 29, 3, 5, 9, 26, 27, 31, 33 or 34, or thecomplement thereof,

[0050] (d) a nucleic acid encoding a protein with an amino acid sequencecomprising the polypeptide as given in SEQ ID NO 32 and which is atleast 70% similar, preferably at least 75%, 80% or 85%, more preferablyat least 90% or 95%, most preferably at least 99% similar to the aminoacid sequence as given in SEQ ID NO 4,

[0051] (e) a nucleic acid encoding a protein with an amino acid sequencewhich is at (east 35% similar, preferably 37%, 40%, 45%, 47% or 50%,similar, more preferably 55%, 60%, 65%, 70%, 75% or 80% similar, mostpreferably 85%, 90% or 95% similar to the amino acid sequence as givenin SEQ ID NO 6,

[0052] (f) a nucleic acid encoding a protein with an amino acid sequencewhich is at least 35% similar, preferably 37%, 40%, 45%, 47% or 50%,similar, more preferably 55%, 60%, 65%, 70%, 75% or 80% similar, mostpreferably 85%, 90% or 95% similar to the amino acid sequence as givenin SEQ ID NO 10 or 35,

[0053] (g) a nucleic acid encoding a protein comprising the amino acidsequence as given in any of SEQ ID NOs 4, 6, 10, 32 or 35,

[0054] (h) a nucleic acid which is degenerated to a nucleic acid asgiven in any of SEQ ID NOs 29, 3, 5, 9, 26, 27, 33 or 34 or which isdegenerated to a nucleic acid as defined in any of (a) to (g) as aresult of the genetic code,

[0055] (i) a nucleic acid which is diverging from a nucleic acidencoding a protein as given in any of SEQ ID NOs 4, 6, 10 or 35 or whichis diverging from a nucleic acid as defined in any of (a) to (g) due tothe differences in codon usage between the organisms,

[0056] (j) a nucleic acid encoding a protein as given in SEQ ID NOs 4,6, 10 or 35, or a nucleic acid as defined in (a) to (g) which isdiverging due to the differences between alleles,

[0057] (k) a nucleic acid encoding an immunologically active fragment ofa cytokinin oxidase encoded by a nucleic acid as given in any of SEQ IDNOs 29, 3, 5, 9, 26, 27, 31, 33 or 34, or an immunologically activefragment of a nucleic acid as defined in any of (a) to (j),

[0058] (l) a nucleic acid encoding a functional fragment of a cytokininoxidase encoded by a nucleic acid as given in any of SEQ ID NOs 29, 3,5, 9, 26, 27, 31, 33 or 34, or a functional fragment of a nucleic acidas defined in any of (a) to (j), wherein said fragment has thebiological activity of a cytokinin oxidase, and

[0059] (m) a nucleic acid encoding a protein as defined in SEQ ID NO 4,6, 10 or 35,

[0060] provided that said nucleic acid is not the nucleic acid asdeposited under any of the following Genbank accession numbers:AC005917, AB024035, and AC023754

[0061] The invention also relates to an isolated nucleic acid of theinvention which is DNA, cDNA, genomic DNA or synthetic DNA, or RNAwherein T is replaced by U.

[0062] The invention also relates to a nucleic acid molecule of at least15 nucleotides in length hybridizing specifically with or specificallyamplifying a nucleic acid of the invention. According to anotherembodiment, the invention also relates to a vector comprising a nucleicacid of the invention. In a preferred embodiment, said vector is anexpression vector wherein the nucleic acid is operably linked to one ormore control sequences allowing the expression of said sequence inprokaryotic and/or eukaryotic host cells.

[0063] It should be understood that for expression of the cytokininoxidase genes of the invention in monocots, a nucleic acid sequencecorresponding to the cDNA sequence should be used to avoid mis-splicingof introns in monocots. Preferred cDNA sequences to be expressed inmonocots have a nucleic acid sequence as represented in any of SEQ IDNOs 25 to 30 and 34.

[0064] The invention also relates to a host cell containing any of thenucleic acid molecules or vectors of the invention. Said host cell ischosen from the group comprising bacterial, insect, fungal, plant oranimal cells.

[0065] Another embodiment of the invention relates to an isolatedpolypeptide encodable by a nucleic acid of the invention, or a homologueor a derivative thereof, or an immunologically active or a functionalfragment thereof. Preferred polypeptides of the invention comprise theamino acid sequences as represented in any of SEQ ID NOs 2, 4, 6, 8, 10,12, 32 and 35, or a homologue or a derivative thereof, or animmunologically active and/or functional fragment thereof. In an evenmore preferred embodiment, the invention relates to a polypeptide whichhas an amino acid sequence as given in SEQ ID NO 2, 4, 6, 8, 10, 12 or35, or a homologue or a derivative thereof, or an immunologically activeand/or functional fragment thereof. Preferred functional fragmentsthereof are those fragments which are devoid of their signal peptide.

[0066] According to yet another embodiment, the invention relates to amethod for producing a polypeptide of the invention comprising culturinga host cell of the invention under conditions allowing the expression ofthe polypeptide and recovering the produced polypeptide from theculture.

[0067] The invention also relates to an antibody specificallyrecognizing a polypeptide of the invention or a specific epitopethereof.

[0068] The invention further relates to a method for the production oftransgenic plants, plant cells or plant tissues comprising theintroduction of a nucleic acid molecule of the invention in anexpressible format or a vector of the invention in said plant, plantcell or plant tissue.

[0069] The invention also relates to a method for the production ofaltered plants, plant cells or plant tissues comprising the introductionof a polypeptide of the invention directly into a cell, a tissue or anorgan of said plant.

[0070] According to another embodiment, the invention relates to amethod for effecting the expression of a polypeptide of the inventioncomprising the introduction of a nucleic acid molecule of the inventionoperably linked to one or more control sequences or a vector of theinvention stably into the genome of a plant cell. The invention furtherrelates to the method as described above further comprising regeneratinga plant from said plant cell. The invention also relates to a transgenicplant cell comprising a nucleic acid sequence of the invention which isoperably linked to regulatory elements allowing transcription and/orexpression of said nucleic acid in plant cells or obtainable by a methodas explained above.

[0071] According to another preferred embodiment, the invention relatesto a transgenic plant cell as described here above wherein the nucleicacid of the invention is stably integrated into the genome of said plantcell.

[0072] The invention further relates to a transgenic plant or planttissue comprising plant cells as herein described and also to aharvestable part of said transgenic plant, preferably selected from thegroup consisting of seeds, leaves, fruits, stem cultures, roots, tubers,rhizomes and bulbs. The invention also relates to the progeny derivedfrom any of said transgenic plants or plant parts.

[0073] According to another embodiment, the invention relates to amethod for stimulating root growth comprising expression of a nucleicacid of the invention or comprising expression of another protein thatreduces the level of active cytokinins in plants or plant parts.

[0074] A plant cell or tissue culture is an artificially producedculture of plants cells or plant tissues that is grown in a specialmedium, either liquid or solid, which provides these plant cells ortissues with all requirements necessary for growth and/or production ofcertain compounds. Plant cell and/or tissue cultures can be used for therapid propagation of plants and for the production of transgenic plantto name a few examples. Root formation can be difficult for someexplants or under some conditions in said cultures and expression of acytokinin oxidase gene in said cultured plant cells or tissue(s) can beused to enhance root formation. Plant cell and/or tissue culture canalso be used for the industrial production of valuable compounds.Possible production compounds are pharmaceuticals, pesticides, pigments,cosmetics, perfumes, food additives, etc. An example of such a productis shikonin, which is produced by the roots of the plant Lithospermumerythrorhizon. An example of a plant tissue culture is a hairy rootculture, which is an artificially produced mass of hairy roots. Roots ofL. erythrorhizon are difficult to collect in large numbers and bypreparing hairy root cultures, the end product shikonin could beindustrially prepared at a faster rate than would normally occur. Asdisclosed herein, expression of cytokinin oxidases enhances root growthand development and can therefore be used advantageously in said plantcell and tissue culture procedures. Therefore, according to anotherembodiment of this invention, a method is provided for stimulating rootgrowth and development comprising expression of a nucleic acid encodinga plant cytokinin oxidase, preferably a cytokinin oxidase of theinvention, in a transgenic plant cell or tissue culture comprising saidtransgenic plant cells.

[0075] The invention further relates to a method for enhancing theformation of lateral or adventitious roots comprising expression of anucleic acid of the invention or comprising expression of anotherprotein that reduces the level of active cytokinins in plants or plantparts.

[0076] The invention also relates to method for altering root geotropismcomprising altering the expression of a nucleic acid of the invention orcomprising expression of another protein that that reduces the level ofactive cytokinins in plants or plant parts.

[0077] The invention also relates to methods for enhancing early vigorand/or for modifying is root/shoot ratio and/or for improving resistanceto lodging and/or for increasing drought tolerance and/or for promotingin vitro propagation of explants comprising expression of a nucleic acidof the invention comprising expression of another protein that reducesthe level of active cytokinins in plants or plant parts.

[0078] The invention further relates to methods for increasing the rootsize or the size of the root meristem comprising expression of a nucleicacid of the invention or comprising expression of another protein thatreduces the level of active cytokinins in plants or plant parts,preferably in roots.

[0079] According to yet another embodiment, the invention relates to amethod for increasing the size of the shoot meristem comprisingdownregulation of expression of a nucleic acid of the invention,preferably in shoots.

[0080] According to a preferred embodiment the invention relates to amethod for delaying leaf senescence comprising downregulation ofexpression of any of the cytokinin oxidases of the invention in leaves,preferably in senescing leaves. Also the invention relates to a methodfor altering leaf senescence comprising expression of one of thecytokinin oxidases in senescing leaves.

[0081] The invention also relates to methods for increasing leafthickness comprising expression of a nucleic acid of the invention orcomprising expression of another protein that reduces the level ofactive cytokinins in plants or plant parts, preferably in leaves.

[0082] The invention also relates to a method for reducing the vesselsize comprising expression of a nucleic acid of the invention orcomprising expression of another protein that reduces the level ofactive cytokinins in plants or plant parts, preferably in vessels. Theinvention further relates to a method for increasing the vessel sizecomprising downregulation of expression of a nucleic acid of theinvention in plants or plant parts. According to another embodiment, theinvention relates to a method for improving standability of seedlingscomprising expression of a nucleic acid of the invention or comprisingexpression of another protein that reduces the level of activecytokinins in seedlings.

[0083] Furthermore, the invention relates to any of the above describedmethods, said method leading to an increase in yield.

[0084] The invention further relates to any of the methods of theinvention wherein said expression of said nucleic acid occurs under thecontrol of a strong constitutive promoter. In a preferred embodiment theinvention relates to any of the methods of the invention wherein saidexpression of said nucleic acid occurs under the control of a promoterthat is preferentially expressed in roots. In Table 5 a non-exhaustivelist of root specific promoters is included. A preferred promoter to beused in the methods of the invention is the root clavata homologpromoter, having a sequence as given in SEQ ID NO 36.

[0085] According to yet another embodiment, the invention relates to amethod for modifying cell fate and/or modifying plant development and/ormodifying plant morphology and/or modifying plant biochemistry and/ormodifying plant physiology and/or modifying the cell cycle progressionrate comprising the modification of expression in particular cells,tissues or organs of a plant, of a nucleic acid of the invention.

[0086] The invention also relates to a method for obtaining enhancedgrowth, and/or increased yield and/or altered senescence of a plantcell, tissue and/or organ and/or increased frequence of formation oflateral organs in a plant, comprising the ectopic expression of anucleic acid of the invention.

[0087] The invention also relates to a method for promoting andextending cell division activity in cells in adverse growth conditionsand/or in stress, comprising the ectopic expression of a nucleic acidsequence of the invention,

[0088] According to yet another embodiment, the invention relates to amethod for identifying and obtaining proteins interacting with apolypeptide of the invention comprising a screening assay wherein apolypeptide of the invention is used.

[0089] In a more preferred embodiment, the invention relates to a methodfor identifying and obtaining proteins interacting with a polypeptide ofthe invention comprising a two-hybrid screening assay wherein apolypeptide of the invention as a bait and a cDNA library as prey areused.

[0090] The invention further relates to a method for modulating theinteraction between a polypeptide of the invention and interactingprotein partners obtainable by a method as described above.

[0091] In a further embodiment, the invention relates to a method foridentifying and obtaining compounds interacting with a polypeptide ofthe invention comprising the steps of:

[0092] a) providing a two-hybrid system wherein a polypeptide of theinvention and an interacting protein partner obtainable by a method asdescribed above,

[0093] b) interacting said compound with the complex formed by theexpressed polypeptides as defined in a), and,

[0094] c) performing (real-time) measurement of interaction of saidcompound with said polypeptide or the complex formed by the expressedpolypeptides as defined in a).

[0095] The invention further relates to a method for identifyingcompounds or mixtures of compounds which specifically bind to apolypeptide of the invention, comprising:

[0096] a) combining a polypeptide of the invention with said compound ormixtures of compounds under conditions suitable to allow complexformation, and,

[0097] b) detecting complex formation, wherein the presence of a complexidentifies a compound or mixture which specifically binds saidpolypeptide.

[0098] The invention also relates to a method as described above whereinsaid compound or mixture inhibits the activity of said polypeptide ofthe invention and can be used for the rational design of chemicals.

[0099] According to another embodiment, the invention relates to the useof a compound or mixture identified by means of a method as describedabove as a plant growth regulator or herbicide.

[0100] The invention also relates to a method for production of a plantgrowth regulator or herbicide composition comprising the steps of thecompound screening methods described above and formulating the compoundsobtained from said steps in a suitable form for the application inagriculture or plant cell or tissue culture.

[0101] The invention also relates to a method for increasing branchingcomprising expression of a nucleic acid of the invention in plants orplant parts, preferably in stems or axillary buds.

[0102] The invention also relates to a method for improving lodgingresistance comprising expression of a nucleic acid of the invention inplants or plant parts, preferably in stems or axillary buds.

[0103] The invention also relates to a method for the design of orscreening for growth-promoting chemicals or herbicides comprising theuse of a nucleic acid of the invention or a vector of the invention.

[0104] According to another embodiment, the invention relates to the useof a nucleic acid molecule of the invention, a vector of of theinvention or a polypeptide of the invention for increasing yield.

[0105] The invention also relates to the use of a nucleic acid moleculeof of the invention, a vector of the invention or a polypeptide of theinvention for stimulating root growth.

[0106] The invention also relates to the use of a nucleic acid moleculeof the invention, a vector of the invention or a polypeptide of theinvention for enhancing the formation of lateral or adventitious roots.

[0107] The invention also relates to the use of a nucleic acid moleculeof the invention, a vector of of the invention or a polypeptide of theinvention for altering root geotropism.

[0108] The invention further relates to the use of a nucleic acidmolecule of of the invention, a vector of the invention or a polypeptideof the invention for enhancing early vigor and/or for modifyingroot/shoot ratio and/or for improving resistance to lodging and/or forincreasing drought tolerance and/or for promoting in vitro propagationof explants.

[0109] The invention also relates to the use of a nucleic acid moleculeof the invention, a recombinant vector of the invention or a polypeptideof the invention for modifying plant development and/or for modifyingplant morphology and/or for modifying plant biochemistry and/or formodifying plant physiology.

[0110] According to yet another embodiment, the invention relates to adiagnostic composition comprising at least a nucleic acid molecule ofthe invention, a vector of the invention, a polypeptide of the inventionor an antibody of the invention.

[0111] Another embodiment of the current invention relates to the use ofa transgenic rootstock that has an enhanced root growth and developmentdue to expression of a cytokinin oxidase in grafting procedures with ascion to produce a plant or tree with improved agricultural orhorticultural characteristics. The scion may be transgenic ornon-transgenic. Specific characteristics envisaged by this embodimentare those conferred by root systems and include improved anchoring ofthe plant/tree in the soil and/or improved uptake of water resulting forexample in improved drought tolerance, and/or improved nutrient uptakefrom the soil and/or improved transport of organic substances throughoutthe plant and/or enhanced secretion of substances into the soil such asfor example phytosiderophores, and/or improved respiration and/orimproved disease resistance and/or enhanced yield. An advantage of usingAtCKX transformed rootstocks for grafting, in addition to their enhancedroot system, is the delayed senescence of leaves on the graft, asdisclosed herein (see FIG. 12A). Preferred plants or trees for thisparticular embodiment include plants or trees that do not grow well ontheir own roots and are grafted in cultivated settings such ascommercially profitable varieties of grapevines, citrus, apricot,almond, plum, peach, apple, pear, cherry, walnut, fig, hazel and loquat.

[0112] As mentioned supra, auxins and cytokinins act as antagonists incertain biological processes. For example, the cytokinin/auxin ratioregulates the production of roots and shoots with a high concentrationof auxin resulting in organized roots and a high concentration ofcytokinins resulting in shoot production. As disclosed in thisinvention, expression of cytokinin oxidases in tobacco and Arabidopsisresults in enhanced root development consistent with enhanced auxineffects. Auxins are also involved in the development of fruit. Treatmentof female flower parts with auxin results in the development ofparthenocarpic fruit in some plant species. Parthenocarpic fruitdevelopment has been genetically engineered in several horticulturalcrop plants through increased biosynthesis of auxins in the femalereproductive organs (WO0105985). Therefore, according to anotherembodiment, this invention relates to a method for inducing theparthenocarpic trait in plants, said method consisting of downregulatingthe expression of one or more cytokinin oxidases or of another proteinthat reduces the level of active cytokinins in plants or plant parts,preferably in the female reproductive organs such as the placenta,ovules and tissues derived therefrom. The DefH9 promoter region fromAntirrhinum majus or one of its homologues, which confer high expressionspecificity in placenta and ovules, can be used for this purpose.

DEFINITIONS AND ELABORATIONS TO THE EMBODIMENTS

[0113] Those skilled in the art will be aware that the inventiondescribed herein is subject to variations and modifications other thanthose specifically described. It is to be understood that the inventiondescribed herein includes all such variations and modifications. Theinvention also includes all such steps, features, compositions andcompounds referred to or indicated in this specification, individuallyor collectively, and any and all combinations of any or more of saidsteps or features.

[0114] The present invention is applicable to any plant, in particular amonocotyiedonous plants and dicotyledonous plants including a fodder orforage legume, ornamental plant, food crop, tree, or shrub selected fromthe list comprising Acacia spp., Acer spp., Actinidia spp., Aesculusspp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogonspp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer,Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza,Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp,Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroemapubescens, Chaenomeles spp., Cinnamomum cassia, Coffea arabica,Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegusspp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga,Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydoniaoblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp.,Dicksonia squarosa, Diheteropogon amplectens, Dioclea spp, Dolichosspp., Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp., Eleusinecoracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Eucleaschimperi, Eulalia villosa, Fagopyrum spp., Feijoa sellowiana, Fragariaspp., Flemingia spp, Freycinetia banksii, Geranium thunbergii, Ginkgobiloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevilleaspp., Guibourtia coleosperma, Hedysarum spp., Hemarthia altissima,Heteropogon contortus, Hordeum vulgare, Hyparrhenia rufa, Hypericumerectum, Hyperthelia dissoluta, Indigo incarnata, Iris spp., Leptarrhenapyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala,Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare,Malus spp., Manihot esculenta, Medicago sativa, Metasequolaglyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp.,Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp.,Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis,Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisumsativum, Podocarpus totara, Pogonarthia fleckii, Pogonarthria squarrosa,Populus spp., Prosopis cineraria, Pseudotsuga menziesli, Pterolobiumstellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata,Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp.,Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyriumsanguineum, Sciadopitys verticillata, Sequoia sempervirens,Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolusfimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp,Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp.,Tsuga heterophylla, Vaccinium spp., Vicia spp. Vitis vinifera, Watsoniapyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke,asparagus, broccoli, brussel sprout, cabbage, canola, carrot,cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape,okra, onion, potato, rice, soybean, straw, sugarbeet, sugar cane,sunflower, tomato, squash, and tea, amongst others, or the seeds of anyplant specifically named above or a issue, cell or organ culture of anyof the above species.

[0115] Throughout this specification, unless the context requiresotherwise the word “comprise”, and variations such as “comprises” and“comprising”, will be understood to imply the inclusion of a statedinteger or step or group of integers or steps but not the exclusion ofany other integer or step or group of integers or steps.

[0116] As used herein, the term “derived from” shall be taken toIndicate that a particular integer or group of integers has originatedfrom the species specified, but has not necessarily been obtaineddirectly from the specified source.

[0117] The terms “protein(s)”, “peptide(s)” or “oligopeptide(s)”, whenused herein refer to amino acids in a polymeric form of any length. Saidterms also include known amino acid modifications such as disulphidebond formation, cysteinylation, oxidation, glutathionylation,methylation, acetylation, farnesylation, biotinylation, stearoylation,formylation, lipoic acid addition, phosphorylation, sulphation,ubiquitination, myristoylation, palmitoylation, geranylgeranylation,cyclization (e.g. pyroglutamic acid formation), oxidation, deamidation,dehydration, glycosylation (e.g. pentoses, hexosamines,N-acetylhexosamines, deoxyhexoses, hexoses, sialic acid etc.) andacylation as well as non-naturally occurring amino acid residues,L-amino acid residues and D-amino acid residues.

[0118] “Homologues” of a protein of the invention are those peptides,oligopeptides, polypeptides, proteins and enzymes which contain aminoacid substitutions, deletions and/or additions relative to the saidprotein with respect to which they are a homologue, without altering oneor more of its functional properties, in particular without reducing theactivity of the resulting. For example, a homologue of said protein willconsist of a bioactive amino acid sequence variant of said protein. Toproduce such homologues, amino acids present in the said protein can bereplaced by other amino acids having similar properties, for examplehydrophobicity, hydrophilicity, hydrophobic moment, antigenicity,propensity to form or break α-helical structures or β-sheet structures,and so on. An overview of physical and chemical properties of aminoacids is given in Table 1. Substitutional variants of a protein of theinvention are those in which at least one residue in said protein aminoacid sequence has been removed and a different residue inserted in itsplace. Amino acid substitutions are typically of single residues, butmay be clustered depending upon functional constraints placed upon thepolypeptide; insertions will usually be of the order of about 1-10 aminoacid residues, and deletions will range from about 1-20 residues.Preferably, amino acid substitutions will comprise conservative aminoacid substitutions, such as those described supra. TABLE 1 Properties ofnaturally occurring amino acids. Charge properties/ hydrophobicity Sidegroup Amino Acid nonpolar Aliphatic ala, ile, leu, val hydrophobicaliphatic, S-containing met aromatic phe, trp imino pro polar unchargedAliphatic gly amide asn, gln aromatic tyr hydroxyl ser, thr sulfhydrylcys positively charged Basic arg, his, lys negatively charged Acidicasp, glu

[0119] Insertional amino acid sequence variants of a protein of theinvention are those in which one or more amino acid residues areintroduced into a predetermined site in said protein. Insertions cancomprise amino-terminal and/or carboxy-terminal fusions as well asintra-sequence insertions of single or multiple amino acids. Generally,insertions within the amino acid sequence will be smaller than amino orcarboxyl terminal fusions, of the order of about 1 to 10 residues.Examples of amino- or carboxy-terminal fusion proteins or peptidesinclude the binding domain or activation domain of a transcriptionalactivator as used in a two-hybrid system, phage coat proteins,(histidine)₆-tag, glutathione S-transferase, protein A, maltose-bindingprotein, dihydrofolate reductase, Tag•100 epitope (EETARFQPGYRS), c-mycepitope (EQKLISEEDL), FLAG®-epitope (DYKDDDK), lacZ, CMP(calmodulin-binding peptide), HA epitope (YPYDVPDYA), protein C epitope(EDOVDPRLIDGK) and VSV epitope (YTDIEMNRLGK).

[0120] Deletional variants of a protein of the invention arecharacterised by the removal of one or more amino acids from the aminoacid sequence of said protein.

[0121] Amino acid variants of a protein of the invention may readily bemade using peptide synthetic techniques well known in the art, such assolid phase peptide synthesis and the like, or by recombinant DNAmanipulations. The manipulation of DNA sequences to produce variantproteins which manifest as substitutional, insertional or deletionalvariants are well known in the art. For example, techniques for makingsubstitution mutations at predetermined sites in DNA having knownsequence are well known to those skilled in the art, such as by M13mutagenesis, T7-Gen in vitro mutagenesis kit (USB, Cleveland, Ohio),QuickChange Site Directed mutagenesis kit (Stratagene, San Diego,Calif.), PCR-mediated site-directed mutagenesis or other site-directedmutagenesis protocols.

[0122] In the current invention “identity” and/or “similarity”percentages between DNA sequences and/or proteins are calculated usingcomputer programs known in the art such as the DNAstar/MegAlign programsin combination with the Clustal method. “Derivatives” of a protein ofthe invention are those peptides, oligopeptides, polypeptides, proteinsand enzymes which comprise at least about five contiguous amino acidresidues of said polypeptide but which retain the biological activity ofsaid protein. A “derivative” may further comprise additionalnaturally-occurring, altered glycosylated, acylated or non-naturallyoccurring amino acid residues compared to the amino acid sequence of anaturally-occurring form of said polypeptide. Alternatively or inaddition, a derivative may comprise one or more non-amino acidsubstituents compared to the amino acid sequence of anaturally-occurring form of said polypeptide, for example a reportermolecule or other ligand, covalently or non-covalently bound to theamino acid sequence such as, for example, a reporter molecule which isbound thereto to facilitate its detection.

[0123] With “immunologically active” is meant that a molecule orspecific fragments thereof such as specific epitopes or haptens arerecognized by, i.e. bind to antibodies. Specific epitopes may bedetermined using, for example, peptide scanning techniques as describedin Geysen et al. (1996) (Geysen, H. M., Rodda, S. J. and Mason, T. J.(1986). A priori delineation of a peptide which mimics a discontinuousantigenic determinant. Mol. Immunol. 23, 709-715.).

[0124] The term “fragment of a sequence” or “part of a sequence” means atruncated sequence of the original sequence referred to. The truncatedsequence (nucleic acid or protein sequence) can vary widely in length;the minimum size being a sequence of sufficient size to provide asequence with at least a comparable function and/or activity or theoriginal sequence referred to (e.g. “functional fragment”), while themaximum size is not critical. In some applications, the maximum sizeusually is not substantially greater than that required to provide thedesired activity and/or function(s) of the original sequence. Typically,the truncated amino acid sequence will range from about 5 to about 60amino acids in length. More typically, however, the sequence will be amaximum of about 50 amino acids in lenght, preferably a maximum of about60 amino acids. It is usually desirable to select sequences of at leastabout 10, 12 or 15 amino acids, up to a maximum of about 20 or 25 aminoacids.

[0125] Functional fragments can also include those comprising an epitopewhich is specific for the proteins according to the invention. Preferredfunctional fragments have a length of at least, for example, 5, 10, 25,100, 150 or 200 amino acids.

[0126] It should thus be understood that functional fragments can alsobe immunologically active fragments or not.

[0127] In the context of the current invention are embodied homologues,derivatives and/or immunologically active and/or functional fragments ofthe cytokinin oxidases as defined supra. Particularly preferredhomologues, derivatives and/or immunologically active and/or functionalfragments of the cytokinin oxidase proteins which are contemplated foruse in the current invention are derived from plants, more specificallyfrom Arabidopsis thaliana, even more specifically said cytokininoxidases are the Arabidopsis thaliana (At)CKX, or are capable of beingexpressed therein. The present invention clearly contemplates the use offunctional homologues or derivatives and/or immunologically activefragments of the AtCKX proteins and is not to be limited in applicationto the use of a nucleotide sequence encoding one of said AtCKX proteins.

[0128] Any of said proteins, polypeptides, peptides and fragmentsthereof can be produced in a biological system, e.g. a cell culture.Alternatively any of said proteins, polypeptides, peptides and fragmentsthereof can be chemically manufactured e.g. by solid phase peptidesynthesis. Said proteins or fragments thereof can be part of a fusionprotein as is the case in e.g. a two-hybrid assay which enables e.g. theidentification of proteins interacting with a cytokinin oxidaseaccording to the invention.

[0129] The proteins or fragments thereof are furthermore useful e.g. tomodulate the interaction between a cytokinin oxidase according to theinvention and interacting protein partners obtained by a method of theinvention. Chemically synthesized peptides are particularly useful e.g.as a source of antigens for the production of antisera and/orantibodies. “Antibodies” include monoclonal, polyclonal, synthetic orheavy chain camel antibodies as well as fragments of antibodies such asFab, Fv or scFv fragments. Monoclonal antibodies can be prepared by thetechniques as described in e.g. Liddle and Cryer (1991) which comprisethe fusion of mouse myeloma cells to spleen cells derived from immunizedanimals. Furthermore, antibodies or fragments thereof to a molecule orfragments thereof can be obtained by using methods as described in e.g.Harlow and Lane (1988). In the case of antibodies directed against smallpeptides such as fragments of a protein of the invention, said peptidesare generally coupled to a carrier protein before immunization ofanimals. Such protein carriers include keyhole limpet hemocyanin (KLH),bovine serum albumin (BSA), ovalbumin and Tetanus toxoid. The carrierprotein enhances the immune response of the animal and provides epitopesfor T-cell receptor binding sites. The term “antibodies” furthermoreincludes derivatives thereof such as labelled antibodies. Antibodylabels include alkaline phosphatase, PKH2, PKH26, PKH67, fluorescein(FITC), Hoechst 33258, R-phycoerythrin (PE), rhodamine (TRITC), QuantumRed, Texas Red, Cy3, biotin, agarose, peroxidase and gold spheres. Toolsin molecular biology relying on antibodies against a protein includeprotein gel blot analysis, screening of expression libraries allowinggene identification, protein quantitative methods including ELISA andRIA, immunoaffinity purification of proteins, immunoprecipitation ofproteins (see e.g. Example 6) and immunolocalization. Other uses ofantibodies and especially of peptide antibodies include the study ofproteolytic processing (Loffler et al. 1994, Woulfe et al. 1994),determination of protein active sites (Lerner 1982), the study ofprecursor and post-translational processing (Baron and Baltimore 1982,Lerner et al. 1981, Sernier et al. 1982), identification of proteindomains involved in protein-protein interactions (Murakami et al. 1992)and the study of exon usage in gene expression (Tamura et al. 1991).

[0130] Embodied in the current invention are antibodies specificallyrecognizing a cytokinin oxidase or homologue, derivative or fragmentthereof as defined supra. Preferably said cytokinin oxidase is a plantcytokinin oxidase, more specifically one of the Arabidopsis thalianacytokinin oxidases (AtCKX).

[0131] The terms “gene(s)”, “polynucleotide(s)”, “nucleic acid(s)”,“nucleic acid sequence(s)”, “nucleotide sequence(s)”, or “nucleic acidmolecule(s)”, when used herein refer to nucleotides, eitherribonucleotides or deoxyribonucleotides or a combination of both, in apolymeric form of any length. Said terms furthermore includedouble-stranded and single-stranded DNA and RNA. Said terms also includeknown nucleotide modifications such as methylation, cyclization and‘caps’ and substitution of one or more of the naturally occurringnucleotides with an analog such as inosine. Modifications of nucleotidesinclude the addition of acridine, amine, biotin, cascade blue,cholesterol, Cy3®, Cy5®, Cys5.5® Dabcyl, digoxigenin, dinitrophenyl,Edans, 6-FAM, fluorescein, 3′-glyceryl, HEX, IRD-700, IRD-800, JOE,phosphate psoralen, rhodamine, ROX, thiol (SH), spacers, TAMRA, TET,AMCA-S®, SE, BODIPY®, Marina Blue®, Pacific Blue®, Oregon Green®,Rhodamine Green®, Rhodamine Red®, Rhodol Green® and Texas Red®.Polynucleotide backbone modifications include methylphosphonate,2′-OMe-methylphosphonate RNA, phosphorothiorate, RNA, 2′-OMeRNA. Basemodifications include 2-amino-dA, 2-aminopurine, 3′-(ddA), 3′dA(cordycepin), 7-deaza-dA, 8-Br-dA, 8-oxo-dA, N⁶-Me-dA, abasic site(dSpacer), biotin dT, 2′-OMe-5Me-C, 2′-OMe-propynyl-C, 3′-(5Me-dC),3′-(ddC), 5-Br-dC, 5-I-dC, 5-Me-dC, 5-F-dC, carboxy-dT, convertible dA,convertible dC, convertible dG, convertible dT, convertible dU,7-deaza-dG, 8-Br-dG, 8-oxo-dG, O⁶-Me-dG, S6-DNP-dG, 4-methyl-indole,5-nitroindole, 2′-OMe-inosine, 2′-dl, 0⁶-phenyl-dl, 4-methyl-indole,2′-deoxynebularine, 5-nitroindole, 2-aminopurine, dP (purine analogue),dK (pyrimidine analogue), 3-nitropyrrole, 2-thio-dT, 4-thio-dT,biotin-dT, carboxy-dT, O⁴-Me dT, O⁴-triazol dT, 2′-OMe-propynyl-U,5-Br-dU, 2′-dU, 5-F-dU, 5-l-dU, O⁴-triazol dU. Said terms also encompasspeptide nucleic acids (PNAs), a DNA analogue in which the backbone is apseudopeptide consisting of N-(2-aminoethyl)-glycine units rather than asugar. PNAs mimic the behaviour of DNA and bind complementary nucleicacid strands. The neutral backbone of PNA results in stronger bindingand greater specificity than normally achieved. In addition, the uniquechemical, physical and biological properties of PNA have been exploitedto produce powerful blomolecular tools, antisense and antigene agents,molecular probes and biosensors.

[0132] The present invention also advantageously provides nucleic acidsequences of at least approximately 15 contiguous nucleotides of anucleic acid according to the invention and preferably from 15 to 50nucleotides. These sequences may, advantageously be used as probes tospecifically hybridise to sequences of the invention as defined above orprimers to initiate specific amplification or replication of sequencesof the invention as defined above, or the like. Such nucleic acidsequences may be produced according to techniques well known in the art,such as by recombinant or synthetic means. They may also be used indiagnostic kits or the like for detecting the presence of a nucleic acidaccording to the invention. These tests generally comprise contactingthe probe with the sample under hybridising conditions and detecting thepresence of any duplex or triplex formation between the probe and anynucleic acid in the sample.

[0133] Advantageously, the nucleic acid sequences, according to theinvention may be produced using such recombinant or synthetic means,such as for example using PCR cloning mechanisms which generally involvemaking a pair of primers, which may be from approximately 15 to 50nucleotides to a region of the gene which is desired to be cloned,bringing the primers into contact with mRNA, cDNA or genomic DNA from acell, performing a polymerase chain reaction under conditions whichbring about amplification of the desired region, isolating the amplifiedregion or fragment and recovering the amplified DNA. Generally, suchtechniques as defined herein are well known in the art, such asdescribed in Sambrook et al. (Molecular Cloning: a Laboratory Manual,1989).

[0134] A “coding sequence” or “open reading frame” or “ORF” is definedas a nucleotide sequence that can be transcribed into mRNA and/ortranslated into a polypeptide when placed under the control ofappropriate control sequences or regulatory sequences, i.e. when saidcoding sequence or ORF is present in an expressible format. Said codingsequence of ORF is bounded by a 5′ translation start codon and a 3′translation stop codon. A coding sequence or ORF can include, but is notlimited to RNA, mRNA, cDNA, recombinant nucleotide sequences,synthetically manufactured nucleotide sequences or genomic DNA. Saidcoding sequence or ORF can be interrupted by intervening nucleic acidsequences.

[0135] Genes and coding sequences essentially encoding the same proteinbut isolated from different sources can consist of substantiallydivergent nucleic acid sequences. Reciprocally, substantially divergentnucleic acid sequences can be designed to effect expression ofessentially the same protein. Said nucleic acid sequences are the resultof e.g. the existence of different alleles of a given gene, of thedegeneracy of the genetic code or of differences in codon usage. Thus,as indicated in Table 2, amino acids such as methionine and tryptophanare encoded by a single codon whereas other amino acids such asarginine, leucine and serine can each be translated from up to sixdifferent codons. Differences in preferred codon usage are illustratedin Table 3 for Agrobacterium tumefaciens (a bacterium), A. thaliana, M.sativa (two dicotyledonous plants) and Oryza sativa (a monocotyledonousplant). To extract one example, the codon GGC (for glycine) is the mostfrequently used codon in A. tumefaciens (36.2%0), is the second mostfrequently used codon in O. sativa but is used at much lower frequenciesin A. thaliana and M. sativa (9% and 8.4% respectively). Of the fourpossible codons encoding glycine (see Table 2), said GGC codon is mostpreferably used in A. tumefaciens and O. sativa. However, in A. thalianathis is the GGA (and GGU) codon whereas in M. sativa this is the GGU(and GGA) codon.

[0136] DNA sequences as defined in the current invention can beinterrupted by intervening sequences. With “intervening sequences” ismeant any nucleic acid sequence which disrupts a coding sequencecomprising said inventive DNA sequence or which disrupts the expressibleformat of a DNA sequence comprising said inventive DNA sequence. Removalof the intervening sequence restores said coding sequence or saidexpressible format. Examples of intervening sequences include intronsand mobilizable DNA sequences such as transposons. With “mobilizable DNAsequence” is meant any DNA sequence that can be mobilized as the resultof a recombination event. TABLE 2 Degeneracy of the genetic code. Three-One- letter letter Amino Acid code code Possible codons Alanine Ala AGCA GCC GCG GCU Arginine Arg R AGA AGG CGA CGC CGG CGU Asparagine Asn NAAC AAU Aspartic Acid Asp D GAC GAU Cysteine Cys C UGC UGU Glutamic AcidGlu E GAA GAG Glutamine Gln Q CAA CAG Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Leucine Leu L UUAUUG CUA CUC CUG CUU Lysine Lys K AAA AAG Methionine Met M AUGPhenylalanine Phe F UUC UUU Proline Pro P CCA CCC CCG CCU Serine Ser SAGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Tryptophan Trp WUGG Tyrosine Tyr Y UAC UAU Valine Val V GUA GUC GUG GUU Possible “STOP”codons UAA UAG UGA

[0137] TABLE 3 Usage of the indicated codons in the different organismsgiven as frequency per thousand codons (http://www.kazusa.or.jp/codon).Agrobacterium Arabidopsis Medicago Oryza Codon tumefaciens thalianasativa sativa UUU 13.9 22.5 24.1 11.3 UUC 24.3 20.7 16.9 26.3 UUA 3.512.9 10.4 4.7 UUG 13.2 21.0 22.4 11.8 UCU 7.0 24.6 19.8 10.1 UCC 14.810.8 7.7 16.9 UCA 7.4 17.8 17.2 9.7 UCG 18.2 8.9 3.2 10.8 UAU 12.3 15.216.6 9.2 UAC 10.3 13.7 14.0 20.6 UAA 0.9 0.9 1.2 0.9 UAG 0.6 0.5 0.8 0.8UGU 3.0 10.8 10.6 5.0 UGC 7.4 7.2 5.8 14.3 UGA 1.8 1.0 0.8 1.3 UGG 12.212.7 10.0 12.8 CUU 19.1 24.3 28.3 14.6 CUC 25.7 15.9 12.0 28.0 CUA 5.210.0 8.8 5.7 CUG 31.6 9.9 8.5 22.1 CCU 7.7 18.3 23.2 11.8 CCC 10.6 5.35.3 12.5 CCA 8.9 16.1 22.6 12.2 CCG 20.7 8.3 3.6 16.7 CAU 10.6 14.0 14.69.2 CAC 9.1 8.7 9.1 14.6 CAA 11.2 19.7 23.2 11.9 CAG 24.9 15.2 12.3 24.6CGU 12.2 8.9 10.1 6.8 CGC 25.5 3.7 4.2 15.9 CGA 8.2 6.2 4.2 4.2 CGG 13.24.8 1.8 9.7 AUU 15.4 22.0 29.4 13.8 AUC 36.9 18.5 14.7 25.5 AUA 6.2 12.911.7 7.2 AUG 24.7 24.5 21.7 24.4 ACU 6.4 17.8 20.8 10.3 ACC 20.9 10.311.7 18.6 ACA 9.1 15.9 18.9 10.0 ACG 18.8 7.6 2.8 10.8 AAU 13.5 22.725.0 12.9 AAC 18.7 20.9 18.7 25.1 AAA 13.6 31.0 32.2 12.0 AAG 24.4 32.635.1 39.4 AGU 5.7 14.0 12.6 7.3 AGC 15.8 11.1 8.8 16.9 AGA 5.3 18.7 13.67.7 AGG 6.5 10.9 11.7 14.9 GUU 16.6 27.3 34.7 15.0 GUC 29.3 12.7 9.922.8 GUA 6.1 10.1 10.0 5.7 GUG 19.7 17.5 16.5 25.0 GCU 17.4 28.0 34.619.8 GCC 35.8 10.3 11.4 33.2 GCA 19.5 17.6 25.9 15.6 GCG 31.7 8.8 3.425.3 GAU 25.8 36.8 40.0 21.5 GAC 28.0 17.3 15.5 31.6 GAA 29.9 34.4 35.917.1 GAG 26.3 32.2 27.4 41.1 GGU 16.5 22.2 28.7 16.3 GGC 36.2 9.0 8.434.7 GGA 12.5 23.9 27.3 15.0 GGG 11.3 10.2 7.4 16.6

[0138] “Hybridization” is the process wherein substantially homologouscomplementary nucleotide sequences anneal to each other. Thehybridization process can occur entirely in solution, i.e. bothcomplementary nucleic acids are in solution. Tools in molecular biologyrelying on such a process include PCR, subtractive hybridization and DNAsequence determination. The hybridization process can also occur withone of the complementary nucleic acids immobilized to a matrix such asmagnetic beads, Sepharose beads or any other resin. Tools in molecularbiology relying on such a process include the isolation of poly (A+)mRNA. The hybridization process can furthermore occur with one of thecomplementary nucleic acids immobilized to a solid support such as anitrocellulose or nylon membrane or immobilized by e.g. photolitographyto e.g. a silicious glass support (the latter known as nucleic acidarrays or microarrays or as nucleic acid chips). Tools in molecularbiology relying on such a process include RNA and DNA gel blot analysis,colony hybridization, plaque hybridization and microarray hybridization.In order to allow hybridization to occur, the nucleic acid molecules aregenerally thermally or chemically (e.g. by NaOH) denatured to melt adouble strand into two single strands and/or to remove hairpins or othersecondary structures from single stranded nucleic acids. The stringencyof hybridization is influenced by conditions such as temperature, saltconcentration and hybridization buffer composition. High stringencyconditions for hybridization include high temperature and/or low saltconcentration (salts include NaCl and Na3-citrate) and/or the inclusionof formamide in the hybridization buffer and/or lowering theconcentration of compounds such as SDS (detergent) in the hybridizationbuffer and/or exclusion of compounds such as dextran sulfate orpolyethylene glycol (promoting molecular crowding) from thehybridization buffer. Conventional hybridization conditions aredescribed in e.g. Sambrook et al. (1989) but the skilled craftsman willappreciate that numerous different hybridization conditions can bedesigned in function of the known or the expected homology and/or lengthof the nucleic acid sequence. Sufficiently low stringency hybridizationconditions are particularly preferred to isolate nucleic acidsheterologous to the DNA sequences of the invention defined supra.Elements contributing to said heterology include allelism, degenerationof the genetic code and differences in preferred codon usage asdiscussed supra.

[0139] Clearly, the current invention embodies the use of the inventiveDNA sequences encoding a cytokinin oxidase, homologue, derivative orimmunologically active and/or functional fragment thereof as definedhigher in any method of hybridization. The current invention furthermorealso relates to DNA sequences hybridizing to said inventive DNAsequences. Preferably said cytokinin oxidase is a plant cytokininoxidase, more specifically the Arabidopsis thaliana (At)CKX.

[0140] To effect expression of a protein in a cell, tissue or organ,preferably of plant origin, either the protein may be introduceddirectly to said cell, such as by microinjection or ballistic means oralternatively, an isolated nucleic acid molecule encoding said proteinmay be introduced into said cell, tissue or organ in an expressibleformat.

[0141] Preferably, the DNA sequence of the invention comprises a codingsequence or open reading frame (ORF) encoding a cytokinin oxidaseprotein or a homologue or derivative thereof or an immunologicallyactive and/or functional fragment thereof as defined supra. Thepreferred protein of the invention comprises the amino acid sequence ofsaid cytokinin oxidase. Preferably said cytokinin oxidase is a plantcytokinin oxidase and more specifically a Arabidopsis thaliana (At)CKX.

[0142] With “vector” or “vector sequence” is meant a DNA sequence whichcan be introduced in an organism by transformation and can be stablymaintained in said organism. Vector maintenance is possible in e.g.cultures of Escherichia coli, A. tumefaciens, Saccharomyces cerevisiaeor Schizosaccharomyces pombe. Other vectors such as phagemids and cosmidvectors can be maintained and multiplied in bacteria and/or viruses.Vector sequences generally comprise a set of unique sites recognized byrestriction enzymes, the multiple cloning site (MCS), wherein one ormore non-vector sequence(s) can be inserted.

[0143] With “non-vector sequence” is accordingly meant a DNA sequencewhich is integrated in one or more of the sites of the MCS comprisedwithin a vector.

[0144] “Expression vectors” form a subset of vectors which, by virtue ofcomprising the appropriate regulatory or control sequences enable thecreation of an expressible format for the inserted non-vectorsequence(s), thus allowing expression of the protein encoded by saidnon-vector sequence(s). Expression vectors are known in the art enablingprotein expression in organisms including bacteria (e.g. E. coli), fungi(e.g. S. cerevisiae, S. pombe, Pichia pastoris), insect cells (e.g.baculoviral expression vectors), animal cells (e.g. COS or CHO cells)and plant cells (e.g. potato virus X-based expression vectors). Thecurrent invention clearly includes any cytokinin oxidase, homologue,derivative and/or immunologically active and/or functional fragmentthereof as defined supra. Preferably said cytokinin oxidase is a plantcytokinin oxidase, more specifically a Arabidopsis thaliana (At)CKX.

[0145] As an alternative to expression vector-mediated proteinproduction in biological systems, chemical protein synthesis can beapplied. Synthetic peptides can be manufactured in solution phase or insolid phase. Solid phase peptide synthesis (Merrifield 1963) is,however, the most common way and involves the sequential addition ofamino acids to create a linear peptide chain. Solid phase peptidesynthesis includes cycles consisting of three steps: (i) immobilizationof the carboxy-terminal amino acid of the growing peptide chain to asolid support or resin; (ii) chain assembly, a process consisting ofactivation, coupling and deprotection of the amino acid to be added tothe growing peptide chain; and (iii) cleavage involving removal of thecompleted peptide chain from the resin and removal of the protectinggroups from the amino acid side chains. Common approaches in solid phasepeptide synthesis include Fmoc/tBu(9-fluorenylmethyloxycarbonyl/t-butyl) and Boc (t-butyloxycarbonyl) asthe amino-terminal protecting groups of amino acids. Amino acid sidechain protecting groups include methyl (Me), formyl (CHO), ethyl (Et),acetyl (Ac), t-butyl (t-Bu), anisyl, benzyl (Bzl), trifluroacetyl (Tfa),N-hydroxysuccinimide (ONSu, OSu), benzoyl (Bz), 4-methylbenzyl (Meb),thioanizyl, thiocresyl, benzyloxymethyl (Bom), 4-nitrophenyl (ONp),benzyloxycarbonyl (Z), 2-nitrobenzoyl (NBz), 2-nitrophenylsulphenyl(Nps), 4-toluenesulphonyl (Tosyl,Tos), pentafluorophenyl (Pfp),diphenylmethyl (Dpm), 2-chlorobenzyloxycarbonyl (Cl-Z),2,4,5-trichlorophenyl, 2-bromobenzyloxycarbonyl (Br-Z), tripheylmethyl(Trityl, Trt), and 2,5,7,8-pentamethyl-chroman-6-sulphonyl (Pmc). Duringchain assembly, Fmoc or Boc are removed resulting in an activatedamino-terminus of the amino acid residue bound to the growing chain. Thecarboxy-terminus of the incoming amino acid is activated by conversioninto a highly reactive ester, e.g. by HBTU. With current technologies(e.g. PerSeptive Biosystems 9050 synthesizer, Applied Biosystems Model431A Peptide Synthesizer), linear peptides of up to 50 residues can bemanufactured. A number of guidelines is available to produce peptidesthat are suitable for use in biological systems including (i) limitingthe use of difficult amino acids such as cys, met, trp (easily oxidizedand/or degraded during peptide synthesis) or arg; (ii) minimizehydrophobic amino acids (can impair peptide solubility); and (iii)prevent an amino-terminal glutamic acid (can cyclize to pyroglutamate).

[0146] By “expressible format” is meant that the isolated nucleic acidmolecule is in a form suitable for being transcribed into mRNA and/ortranslated to produce a protein, either constitutively or followinginduction by an intracellular or extracellular signal, such as anenvironmental stimulus or stress (mitogens, anoxia, hypoxia,temperature, salt, light, dehydration, etc) or a chemical compound suchas IPTG (isopropyl-β-D-thiogalactopyranoside) or such as an antibiotic(tetracycline, ampicillin, rifampicin, kanamycin), hormone (e.g.gibberellin, auxin, cytokinin, glucocorticoid, brassinosteroid,ethylene, abscisic acid etc), hormone analogue (indolacefic acid (IAA),2,4-D, etc), metal (zinc, copper, iron, etc), or dexamethasone, amongstothers. As will be known to those skilled in the art, expression of afunctional protein may also require one or more post-translationalmodifications, such as glycosylation, phosphorylation,dephosphorylation, or one or more protein-protein interactions, amongstothers. All such processes are included within the scope of the term“expressible format”.

[0147] Preferably, expression of a protein in a specific cell, tissue,or organ, preferably of plant origin, is effected by introducing andexpressing an isolated nucleic acid molecule encoding said protein, suchas a cDNA molecule, genomic gene, synthetic oligonucleotide molecule,mRNA molecule or open reading frame, to said cell, tissue or organ,wherein said nucleic acid molecule is placed operably in connection withsuitable regulatory or control sequences including a promoter,preferably a plant-expressible promoter, and a terminator sequence.

[0148] Reference herein to a “promoter” is to be taken in its broadestcontext and includes the transcriptional regulatory sequences derivedfrom a classical eukaryotic genomic gene, including the TATA box whichis required for accurate transcription initiation, with or without aCCAAT box sequence and additional regulatory or control elements (i.e.upstream activating sequences, enhancers and silencers) which alter geneexpression in response to developmental and/or external stimuli, or in atissue-specific manner.

[0149] The term “promoter” also includes the transcriptional regulatorysequences of a classical prokaryotic gene, in which case it may includea −35 box sequence and/or a −10 box transcriptional regulatorysequences.

[0150] The term “promoter” is also used to describe a synthetic orfusion molecule, or derivative which confers, activates or enhancesexpression of a nucleic acid molecule in a cell, tissue or organ.

[0151] Promoters may contain additional copies of one or more specificregulatory elements, to further enhance expression and/or to alter thespatial expression and/or temporal expression of a nucleic acid moleculeto which it is operably connected. Such regulatory elements may beplaced adjacent to a heterologous promoter sequence to drive expressionof a nucleic acid molecule in response to e.g. copper, glucocorticoids,dexamethasone, tetracycline, gibberellin, cAMP, abscisic acid, auxin,wounding, ethylene, jasmonate or salicylic acid or to confer expressionof a nucleic acid molecule to specific cells, tissues or organs such asmeristems, leaves, roots, embryo, flowers, seeds or fruits.

[0152] In the context of the present invention, the promoter preferablyis a plant-expressible promoter sequence. Promoters that also functionor solely function in non-plant cells such as bacteria, yeast cells,insect cells and animal cells are not excluded from the invention. By“plant-expressible” is meant that the promoter sequence, including anyadditional regulatory elements added thereto or contained therein, is atleast capable of inducing, conferring, activating or enhancingexpression in a plant cell, tissue or organ, preferably amonocotyledonous or dicotyledonous plant cell, tissue, or organ.

[0153] The terms “plant-operable” and “operable in a plant” when usedherein, in respect of a promoter sequence, shall be taken to beequivalent to a plant-expressible promoter sequence.

[0154] Regulatable promoters as part of a binary viral plant expressionsystem are also known to the skilled artisan (Yadav 1999—WO9922003;Yadav 2000—WO0017365).

[0155] In the present context, a “regulatable promoter sequence” is apromoter that is capable of conferring expression on a structural genein a particular cell, tissue, or organ or group of cells, tissues ororgans of a plant, optionally under specific conditions, however doesgenerally not confer expression throughout the plant under allconditions. Accordingly, a regulatable promoter sequence may be apromoter sequence that confers expression on a gene to which it isoperably connected in a particular location within the plant oralternatively, throughout the plant under a specific set of conditions,such as following induction of gene expression by a chemical compound orother elicitor.

[0156] Preferably, the regulatable promoter used in the performance ofthe present invention confers expression in a specific location withinthe plant, either constitutively or following induction, however not inthe whole plant under any circumstances. Included within the scope ofsuch promoters are cell-specific promoter sequences, tissue-specificpromoter sequences, organ-specific promoter sequences, cell cyclespecific gene promoter sequences, inducible promoter sequences andconstitutive promoter sequences that have been modified to conferexpression in a particular part of the plant at any one time, such as byintegration of said constitutive promoter within a transposable geneticelement (Ac, Ds, Spm, En, or other transposon).

[0157] Similarly, the term “tissue-specific” shall be taken to indicatethat expression is predominantly in a particular tissue or tissue-type,preferably of plant origin, albeit not necessarily exclusively in saidtissue or tissue-type.

[0158] Similarly, the term “organ-specific” shall be taken to indicatethat expression is predominantly in a particular organ, preferably ofplant origin, albeit not necessarily exclusively in said organ.

[0159] Similarly, the term “cell cycle specific” shall be taken toindicate that expression is predominantly cyclic and occurring in one ormore, not necessarily consecutive phases of the cell cycle albeit notnecessarily exclusively in cycling cells, preferably of plant origin.

[0160] Those skilled in the art will be aware that an “induciblepromoter” is a promoter the transcriptional activity of which isincreased or induced in response to a developmental, chemical,environmental, or physical stimulus. Similarly, the skilled craftsmanwill understand that a “constitutive promoter” is a promoter that istranscriptionally active throughout most, but not necessarily all partsof an organism, preferably a plant, during most, but not neccessarilyall phases of its growth and development.

[0161] Those skilled in the art will readily be capable of selectingappropriate promoter sequences for use in regulating appropriateexpression of the cytokinin oxidase protein from publicly-available orreadily-available sources, without undue experimentation. Placing anucleic acid molecule under the regulatory control of a promotersequence, or in operable connection with a promoter sequence, meanspositioning said nucleic acid molecule such that expression iscontrolled by the promoter sequence. A promoter is usually, but notnecessarily, positioned upstream, or at the 5′-end, and within 2 kb ofthe start site of transcription, of the nucleic acid molecule which itregulates. In the construction of heterologous promoter/structural genecombinations it is generally preferred to position the promoter at adistance from the gene transcription start site that is approximatelythe same as the distance between that promoter and the gene it controlsin its natural setting (i.e., the gene from which the promoter isderived). As is known in the art, some variation in this distance can beaccommodated without loss of promoter function. Similarly, the preferredpositioning of a regulatory sequence element with respect to aheterologous gene to be placed under its control is defined by thepositioning of the element in its natural setting (i.e., the gene fromwhich it is derived). Again, as is known in the art, some variation inthis distance can also occur.

[0162] Examples of promoters suitable for use in gene constructs of thepresent invention include those listed in Table 4, amongst others. Thepromoters listed in Table 4 are provided for the purposes ofexemplification only and the present invention is not to be limited bythe list provided therein. Those skilled in the art will readily be in aposition to provide additional promoters that are useful in performingthe present invention.

[0163] In the case of constitutive promoters or promoters that induceexpression throughout the entire plant, it is preferred that suchsequences are modified by the addition of nucleotide sequences derivedfrom one or more of the tissue-specific promoters listed in Table 4, oralternatively, nucleotide sequences derived from one or more of theabove-mentioned tissue-specific inducible promoters, to confertissue-specificity thereon. For example, the CaMV 35S promoter may bemodified by the addition of maize Adh1 promoter sequence, to conferanaerobically-regulated root-specific expression thereon, as describedpreviously (Ellis et al., 1987). Another example describes conferringroot specific or root abundant gene expression by fusing the CaMV35Spromoter to elements of the maize glycine-rich protein GRP3 gene (Feixand Wulff 2000—WO0015662). Such modifications can be achieved by routineexperimentation by those skilled in the art.

[0164] The term “terminator” refers to a DNA sequence at the end of atranscriptional unit which signals termination of transcription.Terminators are 3′-non-translated DNA sequences containing apolyadenylation signal, which facilitates the addition of polyadenylatesequences to the 3′-end of a primary transcript. Terminators active incells derived from viruses, yeasts, moulds, bacteria, insects, birds,mammals and plants are known and described in the literature. They maybe isolated from bacteria, fungi, viruses, animals and/or plants. TABLE4 Exemplary plant-expressible promoters for use in the performance ofthe present invention I: CELL-SPECIFIC, TISSUE-SPECIFIC, ANDORGAN-SPECIFIC PROMOTERS EXPRESSION GENE SOURCE PATTERN REFERENCEα-amylase (Amy32b) aleurone Lanahan, M.B., et al., Plant Cell 4:203-211, 1992; Skriver, K, et al. Proc. Natl. Acad. Sci. (USA) 88:7266-7270, 1991 cathepsin β-like gene aleurone Cejudo, F. J., et al.,Plant Molecular Biology 20: 849-856, 1992. Agrobacterium cambium Nilssonet al., Physiol. Plant. 100: 456-462, rhizogenes rolB 1997 AtPRP4flowers http://salus.medium.edu/mmg/tierney/html chalcone synthaseflowers Van der Meer, et al., Plant Mol. Biol. (chsA) 15, 95-109, 1990.LAT52 anther Twell et al Mol. Gen Genet. 217: 240-245 (1989) apetala-3flowers chitinase fruit (berries, Thomas et al. CSIRO Plant Industry,grapes, etc) Urrbrae, South Australia, Australia;http://winetitles.com.au/gwrdc/csh95-1.html rbcs-3A green tissue (egLam, E. et al., The Plant Cell 2: 857-866, leaf) 1990.; Tucker et al.,Plant Physiol. 113: 1303-1308, 1992. leaf-specific genes leafBaszczynski, et al., Nucl. Acid Res. 16: 4732, 1988. AtPRP4 leafhttp://salus.medium.edu/mmg/tierney/html chlorella virus adenine leafMitra and Higgins, 1994, Plant methyltransferase gene Molecular Biology26: 85-93 promoter aldP gene promoter leaf Kagaya et al., 1995,Molecular and from rice General Genetics 248: 668-674 rbcs promoter fromrice leaf Kyozuka et al., 1993, Plant or tomato Physiology 102: 991-1000Pinus cab-6 leaf Yamamoto et al., Plant Cell Physiol. 35: 773-778, 1994.rubisco promoter leaf cab (chlorophyll leaf a/b/binding protein SAM22senescent leaf Crowell, et al., Plant Mol. Biol. 18: 459-466, 1992. ltpgene (lipid transfer Fleming, et al, Plant J. 2, 855-862. gene) R.japonicum nif gene Nodule U.S. Pat. No. 4, 803, 165 B. japonicum nifHgene Nodule U.S. Pat. No. 5, 008, 194 GmENOD40 Nodule Yang, et al., ThePlant J. 3: 573-585. PEP carboxylase Nodule Pathirana, et al., PlantMol. Biol. 20: (PEPC) 437-450, 1992. leghaemoglobin (Lb) Nodule Gordon,et al., J. Exp. Bot. 44: 1453-1465, 1993. Tungro bacilliform virusphloem Bhattacharyya-Pakrasi, et al, The gene Plant J. 4: 71-79, 1992.pollen-specific genes pollen; microspore Albani, et al., Plant Mol.Biol. 15: 605, 1990; Albani, et al., Plant Mol. Biol. 16: 501, 1991)Zm13 pollen Guerrero et al Mol. Gen. Genet. 224: 161-168 (1993) apg genemicrospore Twell et al Sex. Plant Reprod. 6: 217-224 (1993) maizepollen-specific pollen Hamilton, et al., Plant Mol. Biol. 18: gene211-218, 1992. sunflower pollen- pollen Baltz, et al., The Plant J. 2:713-721, expressed gene 1992. B. napus pollen- pollen; anther; Arnoldo,et al., J. Cell. Biochem., specific gene tapetum Abstract No. Y101, 204,1992. root-expressible genes roots Tingey, et al., EMBO J. 6: 1, 1987.tobacco auxin-inducible root tip Van der Zaal, et al., Plant Mol. Biol.gene 16, 983, 1991. β-tubulin root Oppenheimer, et al., Gene 63: 87,1988. tobacco root-specific root Conkling, et al., Plant Physiol. 93:genes 1203, 1990. B. napus G1-3b gene root U.S. Pat. No. 5, 401, 836SbPRP1 roots Suzuki et al., Plant Mol. Biol. 21: 109-119 1993. AtPRP1;AtPRP3 roots; root hairs http://salus.medium.edu/mmg/tierney/html RD2gene root cortex http://www2.cnsu.edu/ncsu/research TobRB7 gene rootvasculature http://www2.cnsu.edu/ncsu/research AtPRP4 leaves; flowers;http://salus.medium.edu/mmg/tierney/html lateral root primordiaseed-specific genes seed Simon, et al., Plant Mol. Biol. 5: 191, 1985;Scofield, et al., J. Biol. Chem. 262: 12202, 1987.; Baszczynski, et al.,Plant Mol. Biol. 14: 633, 1990. Brazil Nut albumin seed Pearson, et al.,Plant Mol. Biol. 18: 235-245, 1992. legumin seed Ellis, et al., PlantMol. Biol. 10: 203-214, 1988. glutelin (rice) seed Takaiwa, et al., Mol.Gen. Genet. 208: 15-22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47,1987. zein seed Matzke et al Plant Mol Biol, 14(3): 323-32 1990 napAseed Stalberg, et al, Planta 199: 515-519, 1996. wheat LMW and HMWendosperm Mol Gen Genet 216: 81-90, 1989; glutenin-1 NAR 17: 461-2, 1989wheat SPA seed Albani et al, Plant Cell, 9: 171-184, 1997 wheat α, β,γ-gliadins endosperm EMBO 3: 1409-15, 1984 barley ltr1 promoterendosperm barley B1, C, D, endosperm Theor Appl Gen 98: 1253-62, 1999;hordein Plant J 4: 343-55, 1993; Mol Gen Genet 250: 750-60, 1996 barleyDOF endosperm Mena et al, The Plant Journal, 116(1): 53-62, 1998 blz2endosperm EP99106056.7 synthetic promoter endosperm Vicente-Carbajosa etal., Plant J. 13: 629-640, 1998. rice prolamin NRP33 endosperm Wu et al,Plant Cell Physiology 39(8) 885-889, 1998 rice α-globulin Glb-1endosperm Wu et al, Plant Cell Physiology 39(8) 885-889, 1998 rice OSH1embryo Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122, 1996 riceα-globulin endosperm Nakase et al. Plant Mol. Biol. 33: 513-522REB/OHP-1 1997 rice ADP-glucose PP endosperm Trans Res 6: 157-68, 1997maize ESR gene family endosperm Plant J 12: 235-46, 1997 sorgumγ-kafirin endosperm PMB 32: 1029-35, 1996 KNOX embryo Postma-Haarsma etal, Plant Mol. Biol. 39: 257-71, 1999 rice oleosin embryo and aleuron Wuet at, J. Biochem., 123: 386, 1998 sunflower oleosin seed (embryo andCummins, et al., Plant Mol. Biol. 19: dry seed) 873-876, 1992 LEAFYshoot meristem Weigel et al., Cell 69: 843-859, 1992. Arabidopsisthaliana shoot meristem Accession number AJ131822 knat1 Malus domesticakn1 shoot meristem Accession number Z71981 CLAVATA1 shoot meristemAccession number AF049870 stigma-specific genes stigma Nasrallah, etal., Proc. Natl. Acad. Sci. USA 85: 5551, 1988; Trick, et al., PlantMol. Biol. 15: 203, 1990. class I patatin gene tuber Liu et al., PlantMol. Biol. 153: 386-395, 1991. PCNA rice meristem Kosugi et al, NucleicAcids Research 19: 1571-1576, 1991; Kosugi S. and Ohashi Y, Plant Cell9: 1607-1619, 1997. Pea TubA1 tubulin Dividing cells Stotz and Long,Plant Mol.Biol. 41, 601-614. 1999 Arabidopsis cdc2a cycling cells Chungand Parish, FEBS Lett, 3; 362(2): 215-9, 1995 Arabidopsis Rop1A Anthers;mature Li et al. 1998 Plant Physiol 118, 407-417. pollen + pollen tubesArabidopsis AtDMC1 Meiosis-associated Klimyuk and Jones 1997 Plant J.11, 1-14. Pea PS-IAA4/5 and Auxin-inducible Wong et al. 1996 Plant J. 9,587-599. PS-IAA6 Pea Meristematic Zhou et al. 1997 Plant J. 12, 921-930farnesyltransferase tissues; phloem near growing tissues; light-andsugar-repressed Tobacco (N. sylvestris) Dividing cells/ Trehin et al.1997 Plant Mol.Biol. 35, cyclin B1; 1 meristematic tissue 667-672.Catharanthus roseus Dividing cells/ Ito et al. 1997 Plant J. 11, 983-992Mitotic cyclins CYS (A- meristematic tissue type) and CYM (B-type)Arabidopsis cyc1 At Dividing cells/ Shaul et al. 1996 (=cyc B1; 1) andmeristematic tissue Proc.Natl.Acad.Sci.U.S.A 93, 4868-4872. cyc3aAt(A-type) Arabidopsis tef1 Dividing cells/ Regad et al. 1995Mol.Gen.Genet. promoter box meristematic tissue 248, 703-711.Catharanthus roseus Dividing cells/ Ito et al. 1994 Plant Mol.Biol. 24,863-878. cyc07 meristematic tissue II: EXEMPLARY CONSTITUTIVE PROMOTERSEXPRESSION GENE SOURCE PATTERN REFERENCE Actin constitutive McElroy etal, Plant Cell, 2: 163-171, 1990 CAMV 35S constitutive Odell et al,Nature, 313: 810-812, 1985 CaMV 19S constitutive Nilsson et al.,Physiol. Plant. 100: 456-462, 1997 GOS2 constitutive de Pater et al,Plant J. 2: 837-844, 1992 ubiquitin constitutive Christensen et al,Plant Mol. Biol. 18: 675-689, 1992 rice cyclophilin constitutiveBuchholz et al, Plant Mol Biol. 25: 837-843, 1994 maize histone H3constitutive Lepetit et al, Mol. Gen. Genet. 231: 276-285, 1992 alfalfahistone H3 constitutive Wu et al., Nucleic Acids Res. 17: 3057-3063,1989; Wu et al., Plant Mol. Biol. 11: 641-649, 1988 actin 2 constitutiveAn et al, Plant J. 10(1); 107-121, 1996 III: EXEMPLARY STRESS-INDUCIBLEPROMOTERS NAME STRESS REFERENCE P5CS (delta(1)- salt, water Zhang et al.Plant Science. 129: 81-89, pyrroline-5-carboxylate 1997 syntase) cor15acold Hajela et al., Plant Physiol. 93: 1246-1252, 1990 cor15b coldWlihelm et al., Plant Mol Biol. 23: 1073-1077, 1993 cor15a (−305 to + 78nt) cold, drought Baker et al., Plant Mol Biol. 24: 701-713, 1994 rd29salt, drought, cold Kasuga et al., Nature Biotechnology 18: 287-291,1999 heat shock proteins, heat Barros et al., Plant Mol Biol 19: 665-75,including artificial 1992. Marrs et al., Dev promoters containing Genet14: 27-41, 1993. Schoffl et al., the heat shock element Mol Gen Gent,217: 246-53, 1989. (HSE) smHSP (small heat heat Waters et al, JExperimental Botany shock proteins) 47: 325-338, 1996 wcs120 coldOuellet et al., FEBS Lett. 423: 324-328, 1998 ci7 cold Kirch et al.,Plant Mol Biol 33: 897-909, 1997 Adh cold, drought, hypoxia Dolferus etal., Plant Physiol 105: 1075-87, 1994 pwsi18 water: salt and droughtJoshee et al., Plant Cell Physiol 39: 64-72, 1998 ci21A cold Schneideret al., Plant Physiol 113: 335-45, 1997 Trg-31 drought Chaudhary et al.,Plant Mol Biol 30: 1247-57, 1996 osmotin osmotic Raghothama et al.,Plant Mol Biol 23: 1117-28, 1993 Rab17 osmotic, ABA Vilardell et al.,Plant Mol Biol 17: 985-93, 1991 lapA wounding, enviromental WO99/03977University of California/INRA IV: EXEMPLARY PATHOGEN-INDUCIBLE PROMOTERSNAME PATHOGEN REFERENCE RB7 Root-knot nematodes US5760386 - NorthCarolina State (Meloidogyne spp.) University; Opperman et al (1994)Science 263: 221-23. PR-1, 2, 3, 4, 5, 8, 11 fungal, viral, bacterialWard et al (1991) Plant Cell 3: 1085-1094; Reiss et al 1996; Lebel et al(1998), Plant J, 16(2): 223-33; Melchers et al (1994), Plant J, 5(4):469-80; Lawton et al (1992), Plant Mol Biol, 19(5): 735-43. HMG2nematodes WO9503690 - Virginia Tech Intellectual Properties Inc. Abi3Cyst nematodes Unpublished (Heterodera spp.) ARM1 nematodes Barthels etal., (1997) The Plant Cell 9, 2119-2134. WO 98/31822 —Plant GeneticSystems Att0728 nematodes Barthels et al., (1997) The Plant Cell 9,2119-2134. PCT/EP98/07761 Att1712 nematodes Barthels et al., (1997) ThePlant Cell 9, 2119-2134. PCT/EP98/07761 Gst1 Different types ofStrittmatter et al (1996) Mol. pathogens Plant-Microbe Interact. 9,68-73. LEMMI nematodes WO 92/21757 - Plant Genetic Systems CLEgeminivirus PCT/EP99/03445 - CINESTAV PDF1.2 Fungal Including Manners etal (1998), Plant Mol Altemaria brassicicola Biol, 38(6): 1071-80. andBotrytis cinerea Thi2.1 Fungal - Fusarium Vignutelli et al (1998) Plantoxysporum f sp. J; 14(3): 285-95 matthiolae DB#226 nematodes Bird andWilson (1994) Mol. Plant- Microbe Interact., 7, 419-42 WO 95.322888DB#280 nematodes Bird and Wilson (1994) Mol. Plant- Microbe Interact.,7, 419-42 WO 95.322888 Cat2 nematodes Niebel et at (1995) Mol PlantMicrobe Interact 1995 May-Jun; 8(3): 371-8 □Tub nematodes Aristizabal etal (1996), 8^(th) International Congress on Plant- Microbe Interaction,Knoxville US B-29 SHSP nematodes Fenoll et al (1997) In: Cellular andmolecular aspects of plant- nematode interactions. Kluwer Academic, C.Fenoll, F.M.W. Grundler and S.A. Ohl (Eds.), Tsw12 nematodes Fenoll etal (1997) In: Cellular and molecular aspects of plant- nematodeinteractions. Kluwer Academic, C. Fenoll, F.M.W. Grundler and S. A. Ohl(Eds.) Hs1(pro1) nematodes WO 98/122335 - Jung NsLTP viral, fungal,bacterial Molina & Garc ia-Olmedo (1993) FEBS Lett, 316(2): 119-22 RIPviral, fungal Tumer et al (1997) Proc Natl Acad Sci U.S.A, 94(8):3866-71

[0165] Examples of terminators particularly suitable for use in the geneconstructs of the present invention include the Agrobacteriumtumefaciens nopaline synthase (NOS) gene terminator, the Agrobacteriumtumefaciens octopine synthase (OCS) gene terminator sequence, theCauliflower mosaic virus (CaMV) 35S gene terminator sequence, the Oryzasativa ADP-glucose pyrophosphorylase terminator sequence (t3′Bt2), theZea mays zein gene terminator sequence, the rbcs-1A gene terminator, andthe rbcs-3A gene terminator sequences, amongst others.

[0166] Preferred promoter sequences of the invention include rootspecific promoters such as but not limited to the ones listed in Table 5and as outlined in the Examples. TABLE 5 Exemplary root specificpromoters for use in the performance of the present invention NAMEORIGIN REFERENCE SbPRP1 Soybean Suzuki et al., Plant Mol Biol, 21:109-119, 1993 636 bp fragment of Tobacco Yamamoto et al., Plant CellTobRB7 3: 371-382, 1991 GGPS3 Arabidopsis Okada et al., Plant Physiol122: 1045-1056, 2000 580 bp fragment Arabidopsis Wanapu and Shinmyo, AnnN Y of prxEa Acad Sci 782: 107-114, 1996 Ids2 promoter Barley Okumura etal., Plant Mol Biol 25: 705-719, 1994 AtPRP3 Arabidopsis Fowler et al.,Plant Physiol 121: 1081-1092, 1999

[0167] Those skilled in the art will be aware of additional promotersequences and terminator sequences which may be suitable for use inperforming the invention. Such sequences may readily be used without anyundue experimentation.

[0168] In the context of the current invention, “ectopic expression” or“ectopic overexpression” of a gene or a protein are conferring toexpression patterns and/or expression levels of said gene or proteinnormally not occurring under natural conditions, more specifically ismeant increased expression and/or increased expression levels. Ectopicexpression can be achieved in a number of ways including operablylinking of a coding sequence encoding said protein to an isolatedhomologous or heterologous promoter in order to create a chimeric geneand/or operably linking said coding sequence to its own isolatedpromoter (i.e. the unisolated promoter naturally driving expression atsaid protein) in order to create a recombinant gene duplication or genemultiplication effect. With “ectopic co-expression” is meant the ectopicexpression or ectopic overexpression of two or more genes or proteins.The same or, more preferably, different promoters are used to conferectopic expression of said genes or proteins.

[0169] Preferably, the promoter sequence used in the context of thepresent invention is operably linked to a coding sequence or openreading frame (ORF) encoding a cytokinin oxidase protein or a homologue,derivative or an immunologically active and/or functional fragmentthereof as defined supra.

[0170] “Downregulation of expression” as used herein means loweringlevels of gene expression and/or levels of active gene product and/orlevels of gene product activity. Decreases in expression may beaccomplished by e.g. the addition of coding sequences or parts thereofin a sense orientation (if resulting in co-suppression) or in anantisense orientation relative to a promoter sequence and furthermore bye.g. insertion mutagenesis (e.g. T-DNA insertion or transposoninsertion) or by gene silencing strategies as described by e.g. Angelland Baulcombe (1998—WO9836083), Lowe et al. (1989—WO9853083), Lederer etal. (1999—WO9915682) or Wang et al. (1999-WO9953050). Genetic constructsaimed at silencing gene expression may have the nucleotide sequence ofsaid gene (or one or more parts thereof) contained therein in a senseand/or antisense orientation relative to the promoter sequence. Anothermethod to downregulate gene expression comprises the use of ribozymes.

[0171] Modulating, including lowering, the level of active gene productsor of gene product activity can be achieved by administering or exposingcells, tissues, organs or organisms to said gene product, a homologue,derivative and/or immunologically active fragment thereof.Immunomodulation is another example of a technique capable ofdownregulation levels of active gene product and/or of gene productactivity and comprises administration of or exposing to or expressingantibodies to said gene product to or in cells, tissues, organs ororganisms wherein levels of said gene product and/or gene productactivity are to be modulated. Such antibodies comprise “plantibodies”,single chain antibodies, IgG antibodies and heavy chain camel antibodiesas well as fragments thereof.

[0172] Modulating, including lowering, the level of active gene productsor of gene product activity can futhermore be achieved by administeringor exposing cells, tissues, organs or organisms to an agonist of saidgene product or the activity thereof. Such agonists include proteins(comprising e.g. kinases and proteinases) and chemical compoundsidentified according to the current invention as described supra.

[0173] In the context of the current invention is envisaged thedownregulation of the expression of a cytokinin oxidase gene as definedhigher. Preferably said cytokinin oxidase gene is a plant cytokininoxidase gene, more specifically an AtCKX. The invention furthercomprises downregulation of levels of a cytokinin oxidase protein or ofa cytokinin oxidase activity whereby said cytokinin oxidase protein hasbeen defined supra. Preferably said cytokinin oxidase protein is a plantcytokinin oxidase, more specifically an AtCKX.

[0174] By “modifying cell fate and/or plant development and/or plantmorphology and/or biochemistry and/or physiology” is meant that one ormore developmental and/or morphological and/or biochemical and/orphysiological characteristics of a plant is altered by the performanceof one or more steps pertaining to the invention described herein.

[0175] “Cell fate” refers to the cell-type or cellular characteristicsof a particular cell that are produced during plant development or acellular process therefor, in particular during the cell cycle or as aconsequence of a cell cycle process.

[0176] “Plant development” or the term “plant developmentalcharacteristic” or similar term shall, when used herein, be taken tomean any cellular process of a plant that is involved in determining thedevelopmental fate of a plant cell, in particular the specific tissue ororgan type into which a progenitor cell will develop. Cellular processesrelevant to plant development will be known to those skilled in the art.Such processes include, for example, morphogenesis, photomorphogenesis,shoot development, root development, vegetative development,reproductive development, stem elongation, flowering, and regulatorymechanisms involved in determining cell fate, in particular a process orregulatory process involving the cell cycle.

[0177] “Plant morphology” or the term “plant morphologicalcharacteristic” or similar term will, when used herein, be understood bythose skilled in the art to refer to the external appearance of a plant,including any one or more structural features or combination ofstructural features thereof. Such structural features include the shape,size, number, position, colour, texture, arrangement, and patternationof any cell, tissue or organ or groups of cells, tissues or organs of aplant, including the root, stem, leaf, shoot, petiole, trichome, flower,petal, stigma, style, stamen, pollen, ovule, seed, embryo, endosperm,seed coat, aleurone, fibre, fruit, cambium, wood, heartwood, parenchyma,aerenchyma, sieve element, phloem or vascular tissue, amongst others.

[0178] “Plant biochemistry” or the term “plant biochemicalcharacteristic” or similar term will, when used herein, be understood bythose skilled in the art to refer to the metabolic and catalyticprocesses of a plant, including primary and secondary metabolism and theproducts thereof, including any small molecules, macromolecules orchemical compounds, such as but not limited to starches, sugars,proteins, peptides, enzymes, hormones, growth factors, nucleic acidmolecules, celluloses, hemicelluloses, calloses, lectins, fibres,pigments such as anthocyanins, vitamins, minerals, micronutrients, ormacronutrients, that are produced by plants.

[0179] “Plant physiology” or the term “plant physiologicalcharacteristic” or similar term will, when used herein, be understood torefer to the functional processes of a plant, including developmentalprocesses such as growth, expansion and differentiation, sexualdevelopment, sexual reproduction, seed set, seed development, grainfilling, asexual reproduction, cell division, dormancy, germination,light adaptation, photosynthesis, leaf expansion, fibre production,secondary growth or wood production, amongst others; responses of aplant to externally-applied factors such as metals, chemicals, hormones,growth factors, environment and environmental stress factors (eg.anoxia, hypoxia, high temperature, low temperature, dehydration, light,daylength, flooding, salt, heavy metals, amongst others), includingadaptive responses of plants to said externally-applied factors.

[0180] Means for introducing recombinant DNA into plant tissue or cellsinclude, but are not limited to, transformation using CaCl₂ andvariations thereof, in particular the method described by Hanahan(1983), direct DNA uptake into protoplasts (Krens et al, 1982;Paszkowski et al, 1984), PEG-mediated uptake to protoplasts (Armstronget al, 1990) microparticle bombardment, electroporation (Fromm et al.,1985), microinjection of DNA (Crossway et al, 1986), microparticlebombardment of tissue explants or cells (Christou et al, 1988; Sanford,1988), vacuum-infiltration of tissue with nucleic acid, or in the caseof plants, T-DNA-mediated transfer from Agrobacterium to the planttissue as described essentially by An et al. (1985), Dodds et al.,(1985), Herrera-Estrella et at (1983a, 1983b, 1985). Methods fortransformation of monocotyledonous plants are well known in the art andinclude Agrobacterium-mediated transformation (Cheng et al.,1997—WO9748814; Hansen 1998—WO9854961; Hiel et at, 1994—WO9400977; Hielet at, 1998-WO9817813; Rikllshi et al., 1999—WO9904618; Saito et al,1995—WO9506722), microprojectile bombardment (Adams et al., 1999—U.S.Pat. No. 5,969,213; Bowen et al, 1998—U.S. Pat. No. 5,736,369; Chang etal., 1994—WO9413822; Lundquist et al., 1999—U.S. Pat. No. 5,874,265/U.S.Pat. No. 5,990,390; Vasil and Vasil, 1995—U.S. Pat. No. 5,405,765.Walker et al., 1999—U.S. Pat. No. 5,955,362), DNA uptake (Eyal et al.,1993—WO9318168), microinjection of Agrobacterium cells (von Holt,1994—DE4309203) and sonication (Finer et al., 1997—U.S. Pat. No.5,693,512).

[0181] For microparticle bombardment of cells, a microparticle ispropelled into a cell to produce a transformed cell. Any suitableballistic cell transformation methodology and apparatus can be used inperforming the present invention. Exemplary apparatus and procedures aredisclosed by Stomp et al. (U.S. Pat. No. 5,122,466) and Sanford and Wolf(U.S. Pat. No. 4,945,050). When using ballistic transformationprocedures, the gene construct may incorporate a plasmid capable ofreplicating in the cell to be transformed. Examples of microparticlessuitable for use in such systems include 1 to 5 μm gold spheres. The DNAconstruct may be deposited on the microparticle by any suitabletechnique, such as by precipitation.

[0182] A whole plant may be regenerated from the transformed ortransfected cell, in accordance with procedures well known in the art.Plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a gene constructof the present invention and a whole plant regenerated therefrom. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristem, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand hypocotyl meristem).

[0183] The term “organogenesis”, as used herein, means a process bywhich shoots and roots are developed sequentially from meristematiccentres.

[0184] The term “embryogenesis”, as used herein, means a process bywhich shoots and roots develop together in a concerted fashion (notsequentially), whether from somatic cells or gametes.

[0185] Preferably, the plant is produced according to the inventivemethod is transfected or transformed with a genetic sequence, oramenable to the introduction of a protein, by any art-recognized means,such as microprojectile bombardment, microinjection,Agrobacterium-mediated transformation (including in plantatransformation), protoplast fusion, or electroporation, amongst others.Most preferably said plant is produced by Agrobacterium-mediatedtransformation.

[0186] Agrobacterium-mediated transformation or agrolistictransformation of plants, yeast, moulds or filamentous fungi is based onthe transfer of part of the transformation vector sequences, called theT-DNA, to the nucleus and on integration of said T-DNA in the genome ofsaid eukaryote.

[0187] With “Agrobacterium” is meant a member of the Agrobacteriaceae,more preferably Agrobacterium or Rhizobactedum and most preferablyAgrobacterium tumefaciens.

[0188] With “T-DNA”, or transferred DNA, is meant that part of thetransformation vector flanked by T-DNA borders which is, afteractivation of the Agrobacterium vir genes, nicked at the T-DNA bordersand is transferred as a single stranded DNA to the nucleus of aneukaryotic cell.

[0189] When used herein, with “T-DNA borders”, “T-DNA border region”, or“border region” are meant either right T-DNA border (RB) or left T-DNAborder (LB). Such a border comprises a core sequence flanked by a borderinner region as part of the T-DNA flanking the border and/or a borderouter region as part of the vector backbone flanking the border. Thecore sequences comprise 22 bp in case of octopine-type vectors and 25 bpin case of nopaline-type vectors. The core sequences in the right borderregion and left border region form imperfect repeats. Border coresequences are indispensable for recognition and processing by theAgrobacterium nicking complex consisting of at least VirD1 and VirD2.Core sequences flanking a T-DNA are sufficient to promote transfer ofsaid T-DNA. However, efficiency of transformation using transformationvectors carrying said T-DNA solely flanked by said core sequences islow. Border inner and outer regions are known to modulate efficiency ofT-DNA transfer (Wang et al. 1987). One element enhancing T-DNA transferhas been characterized and resides in the right border outer region andis called overdrive (Peralta et al. 1986, van Haaren et al. 1987).

[0190] With “T-DNA transformation vector” or “T-DNA vector” is meant anyvector encompassing a T-DNA sequence flanked by a right and left T-DNAborder consisting of at least the right and left border core sequences,respectively, and used for transformation of any eukaryotic cell.

[0191] With “T-DNA vector backbone sequence” or “T-DNA vector backbonesequences” is meant all DNA of a T-DNA containing vector that liesoutside of the T-DNA borders and, more specifically, outside the nickingsites of the border core imperfect repeats.

[0192] The current invention includes optimized T-DNA vectors such thatvector backbone integration in the genome of a eukaryotic cell isminimized or absent. With “optimized T-DNA DNA vector” is meant a T-DNAvector designed either to decrease or abolish transfer of vectorbackbone sequences to the genome of a eukaryotic cell. Such T-DNAvectors are known to the one familiar with the art and include thosedescribed by Hanson et al. (1999) and by Stuiver et al.(1999—WO9901563).

[0193] The current invention clearly considers the inclusion of a DNAsequence encoding a cytokinin oxidase, homologue, derivative orimmunologically active and/or functional fragment thereof as definedsupra, in any T-DNA vector comprising binary transformation vectors,super-binary transformation vectors, co-integrate transformationvectors, Ri-derived transformation vectors as well as in T-DNA carryingvectors used in agrolistic transformation. Preferably, said cytokininoxidase is a plant cytokinin oxidase, more specifically an Arabidopsisthaliana (At)CKX.

[0194] With “binary transformation vector” is meant a T-DNAtransformation vector comprising:

[0195] (a) a T-DNA region comprising at least one gene of interestand/or at least one selectable marker active in the eukaryotic cell tobe transformed; and

[0196] (b) a vector backbone region comprising at least origins ofreplication active in E. coli and Agrobacterium and markers forselection in E. coli and Agrobacterium.

[0197] The T-DNA borders of a binary transformation vector can bederived from octopine-type or nopaline-type Ti plasmids or from both.The T-DNA of a binary vector is only transferred to a eukaryotic cell inconjunction with a helper plasmid.

[0198] With “helper plasmid” is meant a plasmid that is stablymaintained in Agrobacterium and is at least carrying the set of virgenes necessary for enabling transfer of the T-DNA. Said set of virgenes can be derived from either octopine-type or nopaline-type Tiplasmids or from both.

[0199] With “super-binary transformation vector” is meant a binarytransformation vector additionally carrying in the vector backboneregion a vir region of the Ti plasmid pTiBo542 of the super-virulent A.tumefaciens strain A281 (EP0604662, EP0687730). Super-binarytransformation vectors are used in conjunction with a helper plasmid.

[0200] With “co-integrate transformation vector” is meant a T-DNA vectorat least comprising:

[0201] (a) a T-DNA region comprising at least one gene of interestand/or at least one selectable marker active in plants; and

[0202] (b) a vector backbone region comprising at least origins ofreplication active in Escherichia coli and Agrobacterium, and markersfor selection in E. coli and Agrobacterium, and a set of vir genesnecessary for enabling transfer of the T-DNA.

[0203] The T-DNA borders and said set of vir genes of a said T-DNAvector can be derived from either octopine-type or nopaline-type Tiplasmids or from both.

[0204] With “Ri-derived plant transformation vector” is meant a binarytransformation vector in which the T-DNA borders are derived from a Tiplasmid and said binary transformation vector being used in conjunctionwith a ‘helper’ Ri-plasmid carrying the necessary set of vir genes.

[0205] As used herein, the term “selectable marker gene” or “selectablemarker” or “marker for selection” includes any gene which confers aphenotype on a cell in which it is expressed to facilitate theidentification and/or selection of cells which are transfected ortransformed with a gene construct of the invention or a derivativethereof. Suitable selectable marker genes contemplated herein includethe ampicillin resistance (Amp′), tetracycline resistance gene (Tc′),bacterial kanamycin resistance gene (Kan′), phosphinothricin resistancegene, neomycin phosphotransferase gene (nptll), hygromycin resistancegene, β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase(CAT) gene, green fluorescent protein (gfp) gene (Haseloff et al, 1997),and luciferase gene, amongst others.

[0206] With “agrolistics”, “agrolistic transformation” or “agrolistictransfer” is meant here a transformation method combining features ofAgrobacterium-mediated transformation and of biolistic DNA delivery. Assuch, a T-DNA containing target plasmid is co-delivered with DNA/RNAenabling in planta production of VirD1 and VirD2 with or without VirE2(Hansen and Chilton 1996; Hansen et al. 1997; Hansen and Chilton1997—WO9712046). With “foreign DNA” is meant any DNA sequence that isintroduced in the host's genome by recombinant techniques. Said foreignDNA includes e.g. a T-DNA sequence or a part thereof such as the T-DNAsequence comprising the selectable marker in an expressible format.Foreign DNA furthermore include intervening DNA sequences as definedsupra. With “recombination event” is meant either a site-specificrecombination event or a recombination event effected by transposon‘jumping’.

[0207] With “recombinase” is meant either a site-specific recombinase ora transposase.

[0208] With “recombination site” is meant either site-specificrecombination sites or transposon border sequences.

[0209] With “site specific recombination event” is meant an eventcatalyzed by a system generally consisting of three elements: a pair ofDNA sequences (the site-specific recombination sequences or sites) and aspecific enzyme (the site-specific recombinase). The site-specificrecombinase catalyzes a recombination reaction only between twosite-specific recombination sequences depending on the orientation ofthe site-specific recombination sequences. Sequences intervening betweentwo site-specific recombination sites will be inverted in the presenceof the site-specific recombinase when the site-specific recombinationsequences are oriented in opposite directions relative to one another(i.e. inverted repeats). If the site-specific recombination sequencesare oriented in the same direction relative to one another (i.e. directrepeats), then any intervening sequences will be deleted uponinteraction with the site-specific recombinase. Thus, if thesite-specific recombination sequences are present as direct repeats atboth ends of a foreign DNA sequence integrated into a eukaryotic genome,such integration of said sequences can subsequently be reversed byinteraction of the site-specific recombination sequences with thecorresponding site specific recombinase. A number of different sitespecific recombinase systems can be used including but not limited tothe Cre/lox system of bacteriophage P1, the FLP/FRT system of yeast, theGin recombinase of phage Mu, the Pin recombinase of E. coli, the PinB,PinD and PinF from Shigelia, and the R/RS system of the pSR1 plasmid.Recombinases generally are integrases, resolvases or flippases. Alsodual-specific recombinases can be used in conjunction with direct orindirect repeats of two different site-specific recombination sitescorresponding to the dual-specific recombinase (WO99/25840). The twopreferred site-specific recombinase systems are the bacteriophage P1Cre/10× and the yeast FLP/FRT systems. In these systems a recombinase(Cre or FLP) interact specifically with its respective site-specificrecombination sequence (lox or FRT respectively) to invert or excise theintervening sequences. The site-specific recombination sequences foreach of these two systems are relatively short (34 bp for lox and 47 bpfor FRT). Some of these systems have already been used with highefficiency in plants such as tobacco (Dale et al. 1990) and Arabidopsis(Osborne et al. 1995). Site-specific recombination systems have manyapplications in plant molecular biology including methods for control ofhomologous recombination (e.g. U.S. Pat. No. 5,527,695), for targetedinsertion, gene stacking, etc. (WO99/25821) and for resolution ofcomplex T-DNA integration patterns or for excision of a selectablemarker (WO99/23202).

[0210] Although the site-specific recombination sequences must be linkedto the ends of the DNA to be excised or to be inverted, the geneencoding the site specific recombinase may be located elsewhere. Forexample, the recombinase gene could already be present in theeukaryote's DNA or could be supplied by a later introduced DNA fragmenteither introduced directly into cells, through crossing or throughcross-pollination. Alternatively, a substantially purified recombinaseprotein could be introduced directly into the eukaryotic cell, e.g. bymicro-injection or particle bombardment. Typically, the site-specificrecombinase coding region will be operably linked to regulatorysequences enabling expression of the site-specific recombinase in theeukaryotic cell.

[0211] With “recombination event effected by transposon jumping” or“transposase-mediated recombination” is meant a recombination eventcatalyzed by a system consisting of three elements: a pair of DNAsequences (the transposon border sequences) and a specific enzyme (thetransposase). The transposase catalyzes a recombination reaction onlybetween two transposon border sequences which are arranged as invertedrepeats. A number of different transposon/transposase systems can beused including but not limited to the Ds/Ac system, the Spm system andthe Mu system. These systems originate from corn but it has been shownthat at least the Ds/Ac and the Spm system also function in other plants(Fedoroff et al. 1993, Schlappi et al. 1993, Van Sluys et al. 1987).Preferred are the Ds- and the Spm-type transposons which are delineatedby 11 bp- and 13 bp-border sequences, respectively.

[0212] Although the transposon border sequences must be linked to theends of the DNA to be excised, the gene encoding the transposase may belocated elsewhere. For example, the recombinase gene could already bepresent in the eukaryote's DNA or could be supplied by a laterintroduced DNA fragment either introduced directly into cells, throughcrossing or through cross-pollination. Alternatively, a substantiallypurified transposase protein could be introduced directly into cells,e.g. by microinjection or by particle bombardment.

[0213] As part of the current invention, transposon border sequences areincluded in a foreign DNA sequence such that they lie outside said DNAsequence and transform said DNA into a transposon-like entity that canmove by the action of a transposase.

[0214] As transposons often reintegrate at another locus of the host'sgenome, segregation of the progeny of the hosts in which the transposasewas allowed to act might be necessary to separate transformed hostscontaining e.g. only the transposon footprint and transformed hostsstill containing the foreign DNA.

[0215] In performing the present invention, the genetic element ispreferably induced to mobilize, such as, for example, by the expressionof a recombinase protein in the cell which contacts the integration siteof the genetic element and facilitates a recombination event therein,excising the genetic element completely, or alternatively, leaving a“footprint”, generally of about 20 nucleotides in length or greater, atthe original integration site. Those hosts and host parts that have beenproduced according to the inventive method can be identified by standardnucleic acid hybridization and/or amplification techniques to detect thepresence of the mobilizable genetic element or a gene constructcomprising the same. Alternatively, in the case of transformed hostcells, tissues, and hosts wherein the mobilizable genetic element hasbeen excised, it is possible to detect a footprint in the genome of thehost which has been left following the excision event, using suchtechniques. As used herein, the term “footprint” shall be taken to referto any derivative of a mobilizable genetic element or gene constructcomprising the same as described herein which is produced by excision,deletion or other removal of the mobilizable genetic element from thegenome of a cell transformed previously with said gene construct. Afootprint generally comprises at least a single copy of therecombination loci or transposon used to promote excision. However, afootprint may comprise additional sequences derived from the geneconstruct, for example nucleotide sequences derived from the left bordersequence, right border sequence, origin of replication,recombinase-encoding or transposase-encoding sequence if used, or othervector-derived nucleotide sequences. Accordingly, a footprint isidentifiable according to the nucleotide sequence of the recombinationlocus or transposon of the gene construct used, such as, for example, asequence of nucleotides corresponding or complementary to a lox site orfit site.

[0216] The term “cell cycle” means the cyclic biochemical and structuralevents associated with growth and with division of cells, and inparticular with the regulation of the replication of DNA and mitosis.Cell cycle includes phases called: G0, Gap1 (G1), DNA synthesis (S),Gap2 (G2), and mitosis (M). Normally these four phases occursequentially, however, the cell cycle also includes modified cycleswherein one or more phases are absent resulting in modified cell cyclesuch as endomitosis, acytokinesis, polyploidy, polyteny, andendoreduplication.

[0217] The term “cell cycle progression” refers to the process ofpassing through the different cell cycle phases. The term “cell cycleprogression rate” accordingly refers to the speed at which said cellcycle phases are run through or the time spans required to complete saidcell cycle phases.

[0218] With “two-hybrid assay” is meant an assay that is based on theobservation that many eukaryotic transcription factors comprise twodomains, a DNA-binding domain (DB) and an activation domain (AD) which,when physically separated (i.e. disruption of the covalent linkage) donot effectuate target gene expression. Two proteins able to interactphysically with one of said proteins fused to DB and the other of saidproteins fused to AD will re-unite the DB and AD domains of thetranscription factor resulting in target gene expression. The targetgene in the yeast two-hybrid assay is usually a reporter gene such asthe β-galactosidase gene. Interaction between protein partners in theyeast two-hybrid assay can thus be quantified by measuring the activityof the reporter gene product (Bartel and Fields 1997). Alternatively, amammalian two-hybrid system can be used which includes e.g. a chimericgreen fluorescent protein encoding reporter gene (Shioda et al, 2000).

[0219] Furthermore, folding simulations and computer redesign ofstructural motifs of the protein of the invention can be performed usingappropriate computer programs (Olszewski, Proteins 25 (1996), 286-299;Hoffman, Comput. Appl. Biosci. 1 (1995), 675-679). Computer modeling ofprotein folding can be used for the conformational and energeticanalysis of detailed peptide and protein models (Monge, J. Mol. Biol.247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45).In particular, the appropriate programs can be used for theidentification of interactive sites of the cytokinin oxidases, itsligands or other interacting proteins by computer assistant searches forcomplementary peptide sequences (Fassina, Immunomethods 5 (1994),114-120). Further appropriate computer systems for the design of proteinand peptides are described in the prior art, for example in Berry,Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann, N.Y. Acac. Sci.501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The resultsobtained form the above-described computer analysis can be used for,e.g. the preparation of peptidomimetics of the protein of the inventionor fragments thereof. Such pseudopeptide analogues of the natural aminoacid sequence of the protein may very efficiently mimic the parentprotein (Benkirane, J. Biol. Chem, 271 (1996), 33218-33224). Forexample, incorporation of easily available achiral Ω-amino acid residuesinto a protein of the invention or a fragment thereof results in thesubstitution of amino bonds by polymethylene units of an aliphaticchain, thereby providing a convenient strategy for constructing apeptidomimetic (Banerjee, Biopolymers 39 (1996), 769-777). Superactivepeptidomimetic analogues of small peptide hormones in other systems aredescribed in the prior art (Zhang, Biochem. Biophys. Res. Commun. 224(1996), 327-331). Appropriate peptidomimetics of the protein of thepresent invention can also be identified by the synthesis ofpeptidomimetic combinatorial libraries through successive aminealkylation and testing the resulting compounds, e.g., for their binding,kinase inhibitory and/or immunlogical properties. Methods for thegeneration and use of peptidomimetic combinatorial libraries aredescribed in the prior art, for example in Ostresh, Methods inEnzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996),709-715.

[0220] Furthermore, a three-dimensional and/or crystallographicstructure of the protein of the invention can be used for the design ofpeptidomimetic inhibitors of the biological activity of the protein ofthe invention (Rose, Biochemistry 35 (1996), 12933-12944; Ruterber,Bioorg. Med. Chem. 4 (1996), 1545-1558).

[0221] The compounds to be obtained or identified in the methods of theinvention can be compounds that are able to bind to any of the nucleicacids, peptides or proteins of the invention. Other interestingcompounds to be identified are compounds that modulate the expression ofthe genes or the proteins of the invention in such a way that either theexpression of said gene or protein is enhanced or decreased by theaction of said compound. Alternatively the compound can exert his actionby enhancing or decreasing the activity of any of the proteins of theinvention. Herein, preferred proteins are novel cytokinin oxidases.

[0222] Said compound or plurality of compounds may be comprised in, forexample, samples, e.g., cell extracts from, e.g., plants, animals ormicroorganisms. Furthermore, said compound(s) may be known in the artbut hitherto not known to be capable of suppressing or activatingcytokinin oxidase interacting proteins. The reaction mixture may be acell free extract of may comprise a cell or tissue culture. Suitable setups for the method of the invention are known to the person skilled inthe art and are, for example, generally described in Alberts et al.,Molecular Biology of the Cell, third edition (1994), in particularChapter 17. The plurality of compounds may be, e.g., added to thereaction mixture, culture medium or injected into the cell.

[0223] If a sample containing a compound or a plurality of compounds isidentified in the method of the invention, then it is either possible toisolate the compound form the original sample identified as containingthe compound capable of acting as an agonist, or one can furthersubdivide the original sample, for example, if it consists of aplurality of different compounds, so as to reduce the number ofdifferent substances per sample and repeat the method with thesubdivisions of the original sample. Depending on the complexity of thesamples, the steps described above can be performed several times,preferably until the sample identified according to the method of theinvention only comprises a limited number of or only one substance(s).Preferably said sample comprises substances or similar chemical and/orphysical properties, and most preferably said substances are identical.Preferably, the compound identified according to the above-describedmethod or its derivative is further formulated in a form suitable forthe application in plant breeding or plant cell and tissue culture.

[0224] The term “early vigor” refers to the ability of a plant to growrapidly during early is development, and relates to the successfulestablishment, after germination, of a well-developed root system and awell-developed photosynthetic apparatus.

[0225] The term “resistance to lodging” or “standability” refers to theability of a plant to fix itself to the soil. For plants with an erector semi-erect growth habit this term also refers to the ability tomaintain an upright position under adverse (environmental) conditions.This trait relates to the size, depth and morphology of the root system.

[0226] The term ‘grafting’ as used herein, refers to the joiningtogether of the parts of two different plants so that they bind togetherand the sap can flow, thus forming a single new plant that can grow anddevelop. A graft therefore consists of two parts: (i) the lower part isthe rootstock as referred to herein and essentially consists of the rootsystem and a portion of the stem, and (ii) the upper part, the scion orgraft, which gives rise to the aerial parts of the plant.

[0227] As used herein, tblastn refers to an alignment tool that is partof the BLAST (Basic Local Alignment Search Tool) family of programs(http://www.ncbi.nlm.nih.gov/BLAST/). BLAST alms to identify regions ofoptimal local alignment, i.e. the alignment of some portion of twonucleic acid or protein sequences, to detect relationships amongsequences which share only isolated regions of similarity (Altschul etal., 1990). In the present invention, tblastn of the BLAST 2.0 suite ofprograms was used to compare the maize cytokinin oxidase proteinsequence against a nucleotide sequence database dynamically translatedin all reading frames (Altschul et al., Nucleic Acids Res. 25: 3389-3402(1997)).

[0228] The following examples and figures are given by means ofillustration of the present invention and are in no way limiting. Thecontents of all references included in this application are incorporatedby reference.

BRIEF DESCRIPTION OF THE FIGURES

[0229]FIG. 1. Schematic representation of plant cytokinin oxidase genes.

[0230] Shown are the structures of different cytokinin oxidase genesisolated from maize (ZmCKX1, accession number AF044603, Biochem.Biophys. Res. Corn. 255:328-333, 1999) and Arabidopsis (AtCKX1 toAtCKX4). Exons are denominated with ‘E’ and represented by shaded boxes.Introns are represented by white boxes. Further indicated are the genesizes (in kb, on top of each structure), the gene accession numbers(under the names) and a size bar representing 0.5 kb.

[0231]FIG. 2. Alignment of plant cytokinin oxidase amino acid sequences.

[0232] The amino acid sequences from cytokinin oxidases from maize(ZmCKX1) and Arabidopsis (AtCKX1 to AtCKX4) are aligned. Identical aminoacid residues are marked by a black box, similar amino acid residues arein a grey box. Amino acid similarity groups: (M,I,L,V), (F,W,Y), (G,A),(S,T), (R,K,H), (E,D), (N,Q),

[0233]FIG. 3. Northern blot analysis of AtCKX1-expressing tobacco andArabidopsis plants.

[0234] (A) Northern blot analysis of constitutively expressing tobaccoplants (lanes 1-8) compared to wild type SNN tobacco (lane 9)

[0235] (B) Comparison of tetracycline-induced gene expression in leavesafter 12 h of induction with a constitutively expressing clone. Lanes2-9, leaves of four different AtCKX1W38TetR clones (+,−, with or withouttetracycline treatment), lane 1, constitutively expressing 35S:: AtCKX1clone.

[0236] (C) Northern blot analysis of Arabidopsis plants constitutivelyexpressing AtCKX1 gene. Lanes 2-4, three different constitutivelyexpressing 35S::AtCKX1 clones compared to wild type Arabidopsis plant(lane 1).

[0237]FIG. 4: Growth characteristics of 35S::AtCKX1 transgenicArabidopsis plants.

[0238] (A) Two wild type seedlings (left) compared to two 35S::AtCKX1expressing seedlings (right). Note the increased formation ofadventitious roots and increased root branching in the trangenicseedlings. Pictures were taken 14 days after germination. Plants weregrown in vitro on MS medium in petri dishes in a vertical position.

[0239] (B) Like A, but roots stained with toluidine blue.

[0240] (C) Top view of a petri dish with 35S::AtCKX1 transgenicseedlings three weeks after germination.

[0241] (D) A 35S::AtCKX1 transgenic plants grown in liquid culture.Roots of wild type seedlings grow poorly under these conditions (notshown).

[0242] (E) Transformants (T0) that express the 35S::AtCKX1 gene (threeplants on the right), a wild type plant is shown on the left.

[0243] (F) Phenotype of T1 plants grown in soil. Wild type plant (left)compared to two 35S::AtCKX1 trangenic plants.

[0244]FIG. 5: Phenotype of AtCKX2 overexpressing Arabidopsis plants.

[0245] T1 generation of 35S::AtCKX2 expressing Arabidopsis plants (twoplants on the right) compared to wild type (plant on the left).

[0246]FIG. 6. Northern blot analysis of AtCKX2 expressing tobacco andArabidopsis plants.

[0247] (A) Northern blot analysis of constitutively expressing tobaccoplants (lanes 1-7) compared to wild type SNN tobacco (lane 8)

[0248] (B) Northern blot analysis of Arabidopsis plants constitutivelyexpressing AtCKX2 gene. Lanes 2-8, seven different consitutivelyexpressing 35S::AtCKX2 clones compared to wild type Arabidopsis plant(lane 1).

[0249]FIG. 7. Shoot phenotype of AtCKX1 and AtCKX2 expressing tobaccoplants.

[0250] (A) Top view of six week old plants.

[0251] (B) Tobacco plants at the flowering stage.

[0252] (C) Kinetics of stem elongation. Arrows mark the onset offlowering. Age of plants (days after germination) and leaf number atthat stage are indicated above the arrows. Bars indicate SD; n=12.

[0253] (D) Number of leaves (n=12) formed between day 68 and day 100after germination and final surface area of these leaves (100% of wildtype is 3646±144 cm²; n=3).

[0254] (E) Comparison of leaf size and senescence. Leaves were fromnodes number 4, 9, 12, 16 and 20 from the top (from left to right).

[0255]FIG. 8. Root phenotype of AtCKX expressing transgenic tobaccoplants.

[0256] (A) Seedlings 17 days after germination.

[0257] (B) Root system of soil grown plants at the flowering stage.

[0258] (C) Root length, number of lateral roots (LR) and adventitiousroots (AR) on day 10 after germination.

[0259] (D) Dose-response curve of root growth Inhibition by exogenouscytokinin. Bars indicate±SD; n=30.

[0260]FIG. 9: Growth of axillary shoot meristems in 35S::AtCKX1expressing, tobacco plants.

[0261]FIG. 10: Histology of shoot meristems, leaves and root meristemsof AtCKX1 overexpressing tobacco plants, versus wild type (WT) tobacco.

[0262] (A) Longitudinal median section through the vegetative shootapical meristem. P. leaf primordia.

[0263] (B) Vascular tissue in second order veins of leaves. X, xylem,PH, a phloem bundle.

[0264] (C) Cross sections of fully developed leaves.

[0265] (D) Scanning electron microscopy of the upper leaf epidermis.

[0266] (E) Root apices stained with DAPI, RM, root meristem.

[0267] (F) Longitudinal median sections of root meristems ten days aftergermination. RC, root cap; PM, promeristem.

[0268] (G) Transverse root sections 10 mm from the apex. E, epidermis,C1-C4, cortical cell layer, X, xylem, PH, phloem. Bars are 100 μm.

[0269]FIG. 11: Northern blot analysis of AtCKX3 and AtCKX4-expressingtobacco plants.

[0270] (A) Northern blot analysis of constitutively expressing AtCKX3tobacco plants. Lane designations indicate individual transgenic plantnumbers, WT is wild type SNN tobacco. The blot on top was probed with aAtCKX3 specific probe, the lower blot with a probe specific for the 25SrRNA and serves as a control for RNA loading.

[0271] (B) Northern blot analysis of constitutively expressing AtCKX4tobacco plants. Lane designations indicate individual transgenic plantnumbers, WT is wild type SNN tobacco. The blot on top was probed with anAtCKX4 specific probe, the lower blot with a probe specific for the 25SrRNA and serves as a control for RNA loading.

[0272]FIG. 12: Recipocal grafts of AtCKX transgenic tobacco plants andwild type plants.

[0273] (A) Two plants on the left: Control (WT scion grafted on a WTrootstock).

[0274] Two plants on the right: WT scion grafted on a AtCKX2-38transgenic rootstock.

[0275] (B) Left: Control (WT scion grafted on a WT rootstock).

[0276] Right: Scion of AtCKX2-38 plant grafted on WT rootstock.

[0277] (C) Magnification of root area.

[0278] Left: Control (WT scion grafted on a WT rootstock).

[0279] Right: WT scion grafted on an AtCKX2-38 transgenic rootstock.

[0280] (D) Formation of adventitious roots.

[0281] Left: Control (WT scion grafted on an WT rootstock).

[0282] Right: WT scion grafted on an AtCKX2-38 transgenic rootstock.

EXAMPLES Example 1 Brief Description of the Sequences of the Invention

[0283] Seq ID No Description 1 AtCKX1 genomic 2 AtCKX1 protein 3 AtCKX2genomic 4 AtCKX2 protein 5 AtCKX3 genomic 6 AtCKX3 protein 7 AtCKX4genomic 8 AtCKX4 protein 9 AtCKX5 genomic (short version) 10 AtCKX5protein (short version) 11 AtCKX6 genomic 12 AtCKX6 protein 13 5′primerAtCKX1 14 3′primer AtCKX1 15 5′primer AtCKX2 16 3′primer AtCKX2 175′primer AtCKX3 18 3′primer AtCKX3 19 5′primer AtCKX4 20 3′primer AtCKX421 5′primer AtCKX5 22 3′primer AtCKX5 23 5′primer AtCKX6 24 3′primerAtCKX6 25 AtCKX1 cDNA 26 AtCKX2 cDNA 27 AtCKX3 cDNA 28 AtCKX4 cDNA 29AtCKX5 cDNA (short version) 30 AtCKX6 cDNA 31 AtCKX2 cDNA fragment 32AtCKX2 peptide fragment 33 AtCKX5 genomic (long version) 34 AtCKX5 cDNA(long version) 35 AtCKX5 protein (long version) 36 root clavata homologpromoter

Example 2 Identification of Candidate Cytokinin Oxidase Encoding Genesfrom Arabidopsis thaliana

[0284] Six different genes were identified from Arabidopsis thalianathat bear sequence similarity to a cytokinin oxidase gene from maize(Morris et al., Biochem Biophys Res Comm 255:328-333, 1999; Houda-Herinet al. Plant J 17:615-626; WO 99/06571). These genes were found byscreening 6-frame translations of nucleotide sequences from publicgenomic databases with the maize protein sequence, employing tblastnprogram. These sequences were designated as Arabidopsis thalianacytokinin oxidase-like genes or AtCKX. They were arbitrarily numbered asAtCKX1 to AtCKX6. The below list summarizes the information on thesegenes. The predicted ORF borders and protein sequences are indicative,in order to Illustrate by approximation the protein sequence divergencebetween the Arabidopsis and maize cytokinin oxidases, as well as amongstthe different Arabidopsis cytokinin oxidases. The ORF borders andprotein sequences shown should not be taken as conclusive evidence forthe mode of action of these AtCKX genes. For DNA and protein sequencecomparisons the program MegAlign from DNAstar was used. This programuses the Clustal method for alignments. For multiple alignments ofprotein and cDNA sequences the gap penalty and gap length penalty wasset at 10 each. For pairwise alignments of proteins the parameters wereas follows: Ktuple at 1; Gap penalty at 3; window at 5; diagonals savedat 5. For pairwise alignments of cDNA's the parameters were as follows:Ktuple at 2; Gap penalty at 5; window at 4; diagonals saved at 4. Thesimilarity groups for protein alignments was: (M,I,L,V), (F,W,Y), (G,A),(S,T), (R,K,H), (E,D), (N,Q). The values that are indicated amongst theArabidopsis cDNA and protein sequences represent the lowest and highestvalues found with all combinations.

[0285] A. Gene name: AtCKX1 (Arabidopsis thaliana cytokinin oxidase-likeprotein 1, SEQ ID NO1)

[0286] Location in database (accession number, location on bac):AC002510, Arabidopsis thaliana chromosome II section 225 of 255 of thecomplete sequence. Sequence from clones T32G6.

[0287] ORF Predicted in the Database:

[0288] 15517 . . . 16183, 16415 . . . 16542, 16631 . . . 16891, 16995 .. . 17257, 17344 . . . 17752

[0289] The AtCKX1 cDNA sequence is listed as SEQ ID NO 25

[0290] Predicted protein sequence: SEQ ID NO 2

[0291] Homologies

[0292] % Identity with Z. mays cDNA:

[0293] 31.5% (Dnastar/MegAlign—Clustal method)

[0294] % Similarity with Z. mays Protein:

[0295] 32.2% (Dnastar/MegAlign—Clustal method)

[0296] % Identity with Other Arabidopsis cDNA's (Range):

[0297] 38.2% (AtCKX2) 54.1% (AtCKX6) (Dnastar/MegAlign—Clustal method)

[0298] % Similarity with Other Arabidopsis Proteins (Range):

[0299] 37.1% (AtCKX2)-58.1% (AtCKX6) (Dnastar/MegAlign—Clustal method)

[0300] B. Gene name: AtCKX2 (Arabidopsis thaliana cytokinin oxidase-likeprotein 2, SEQ ID NO3)

[0301] Location in database (accession number, location on bac):AC005917, Arabidopsis thaliana chromosome II section 113 of 255 of thecomplete sequence. Sequence from clones F27F23, F3P11.

[0302] ORF Predicted in the Database:

[0303] complement, 40721 . . . 41012, 41054 . . . 41364, 41513 . . .41770, 42535 . . . 42662, 43153 . . . 43711

[0304] Please note: The cDNA sequence identified by the inventor usingthe gene prediction program NetPlantGene(http://www.cbs.dtu.dk/services/NetGene2/) was different than the oneannotated in the database. Based on the new cDNA sequence the ORFpredicted in the database was revised:

[0305] complement, 40721 . . . 41012, 41095 . . . 41364, 41513 . . .41770, 42535 . . . 42662, 43153 . . . 43711 The protein sequence encodedby this cDNA is listed as SEQ ID NO 4. The cDNA of AtCKX2 was cloned byRT-PCR from total RNA of AtCKX2 transgenic plant tissue with theone-step RT-PCR kit (Qiagen, Hilden, Germany) and sequenced using an ABIPRISM Big Dye Terminator cycle sequencing reaction kit (Perkin ElmerApplied Biosystems Division). This confirmed that the cDNA sequenceidentified and predicted by the inventor was correct. The new AtCKX2cDNA sequence is listed as SEQ ID NO 26. An 84-bp fragment correspondingto nucleotides 1171 through 1254 of the AtCKX2 cDNA is listed as SEQ IDNO 31. The corresponding peptide sequence of this 84-bp cDNA sequence islisted as SEQ ID NO 32.

[0306] Homologies

[0307] % Identity with Z. mays cDNA:

[0308] 38.4% (Dnastar/MegAlign—Clustal method)

[0309] % Similarity with Z. mays Protein:

[0310] 37.5% (Dnastar/MegAlign—Clustal method)

[0311] % Identity with Other Arabidopsis cDNA's (Range):

[0312] 34.9% (AtCKX6)-64.5% (AtCKX4) (Dnastar/MegAlign—Clustal method)

[0313] % Similarity with Other Arabidopsis Proteins (Range):

[0314] 36.5% (AtCKX6)-66.1% (AtCKX4) (Dnastar/MegAlign—Clustal method)

[0315] C. Gene name: AtCKX3 (Arabidopsis thaliana cytokinin oxidase-likeprotein 3, SEQ ID NO5)

[0316] Location in database (accession number, location on bac):AB024035, Arabidopsis thaliana genomic DNA, chromosome 5, P1 clone:MHM17, complete sequence.

[0317] No prediction of the ORF in the database.

[0318] The gene was identified by the inventor using several geneprediction programs including GRAIL(ftp://arthur.epm.oml.gov/pub/xgrail), Genscan(http://CCR-081.mit.edu/GENSCAN.html) and NetPlantGene(http://www.cbs.dtu.dklservices/NetGene2/):

[0319] complement, 29415 . . . 29718, 29813 . . . 30081, 30183 . . .30443, 30529 . . . 30656, 32107 . . . 32716 The new AtCKX3 cDNA sequenceidentified by the inventor is listed as SEQ ID NO 27

[0320] Predicted protein sequence, based on own ORF prediction: SEQ IDNO 6

[0321] Homologies

[0322] % Identity with Z. mays cDNA:

[0323] 38.7% (Dnastar/MegAlign—Clustal method)

[0324] % Similarity with Z. mays Protein:

[0325] 39.2% (Dnastar/MegAlign—Clustal method)

[0326] % Identity with Other Arabidopsis cDNA's (Range):

[0327] 38.8% (AtCKX6)-51.0% (AtCKX2) (Dnastar/MegAlign—Clustal method)

[0328] % Similarity with Other Arabidopsis Proteins (Range):

[0329] 39.9% (AtCKX6)-46.7% (AtCKX2) (Dnastar/MegAlign—Clustal method)

[0330] D. Gene name: AtCKX4 (Arabidopsis thaliana cytokinin oxidase-likeprotein 4, SEQ ID NO7)

[0331] Location in database (accession number, location on bac):

[0332] 1) AL079344, Arabidopsis thaliana DNA chromosome 4, BAC cloneT16L4 (ESSA project)

[0333] 2) AL161575, Arabidopsis thaliana DNA chromosome 4, contigfragment No. 71.

[0334] ORF Predicted in the Database:

[0335] 1) 76187 . . . 76814, 77189 . . . 77316, 77823 . . . 78080, 78318. . . 78586, 78677 . . . 78968

[0336] 2) 101002 . . . 101629, 102004 . . . 102131, 102638 . . . 102895,103133 . . . 103401, 103492 . . . 103783

[0337] The AtCKX4 cDNA sequence is listed as SEQ ID NO 28

[0338] Predicted protein sequence: SEQ ID NO 8

[0339] Homologies

[0340] % Identity with Z mays cDNA:

[0341] 41.0% (Dnastar/MegAlign—Clustal method)

[0342] % Similarity with Z. mays Protein:

[0343] 41.0% (Dnastar/MegAlign—Clustal method)

[0344] % Identity with Other Arabidopsis cDNA's (Range):

[0345] 35.2% (AtCKX6)-64.5% (AtCKX2) (Dnastar/MegAlign—Clustal method)

[0346] % Similarity with Other Arabidopsis Proteins (Range):

[0347] 35.1% (AtCKX6)-66.1% (AtCKX2) (Dnastar/MegAlign—Clustal method)

[0348] E. Gene name: AtCKX5 (Arabidopsis thaliana cytokinin oxidase-likeprotein 5, SEQ ID NO 9)

[0349] Location in database (accession number, location on bac):AC023754, F1B16, complete sequence, chromosome 1

[0350] No prediction of the ORF in the database.

[0351] The gene was identified by the inventors using several geneprediction programs including GRAIL(ftD://arthur.epm.ornl.cov/pub/xgrail), Genscan(http://CCR-081.mit.edu(GEN SCAN.html) and NetPlantGene(http://www.cbs.dtu.dklservices/NetGene2/).

[0352] 43756 . . . 44347, 44435 . . . 44562, 44700 . . . 44966, 45493 .. . 45755, 46200 . . . 46560

[0353] The new AtCKX5 cDNA sequence identified and predicted by theinventor is listed as SEQ ID NO 29. The predicted protein sequence forthis cDNA is listed as SEQ ID NO 10. A second potential ATG startcodonis present 9 nucleotides more upstream in the genomic sequence. It isunclear which of these 2 startcodons encodes the first amino acid of theprotein. Therefore, a second potential AtCKX5 cDNA starting at thisupstream startcodon is also listed in this invention as SEQ ID NO 34.The corresponding genomic sequence is listed as SEQ ID NO 33 and theencoded protein as SEQ ID NO 35.

[0354] Homologies

[0355] % Identity with Z. mays cDNA:

[0356] 39.1% (Dnastar/MegAlign—Clustal method)

[0357] % Similarity with Z. mays Protein:

[0358] 36.6% (Dnastar/MegAlign—Clustal method)

[0359] % Identity with Other Arabidopsis cDNA's (Range):

[0360] 40.1% (AtCKX2)-44.0% (AtCKX3) (Dnastar/MegAlign—Clustal method)

[0361] % Similarity with Other Arabidopsis Proteins (Range):

[0362] 41.6% (AtCKX4)-46.4% (AtCKX6) (Dnastar/MegAlign—Clustal method)

[0363] F. Gene name: AtCKX6 (Arabidopsis thaliana cytokinin oxidase-likeprotein 6, SEQ ID NO 11)

[0364] Location in database (accession number, location on bac):AL163818, Arabidopsis thaliana DNA chromosome 3, P1 clone MA21 (ESSAproject).

[0365] ORF Predicted in the Database:

[0366] 46630 . . . 47215, 47343 . . . 47470, 47591 . . . 47806, 47899 .. . 48161, 48244 . . . 48565

[0367] The AtCKX6 cDNA sequence is listed as SEQ ID NO 30

[0368] Predicted protein sequence: SEQ ID NO 12

[0369] Homologies

[0370] % Identity with Z. mays cDNA:

[0371] 37.3% (Dnastar/MegAlign—Clustal method)

[0372] % Similarity with Z. mays Protein:

[0373] 36.1% (Dnastar/MegAlign—Clustal method)

[0374] % Identity with Other Arabidopsis cDNA's (Range):

[0375] 34.9% (AtCKX2)-54.1% (AtCKX1) (Dnastar/MegAlign—Clustal method)

[0376] % Similarity with Other Arabidopsis Proteins (Range):

[0377] 35.1% (AtCKX4)-58.1% (AtCKX1) (Dnastar/MegAlign—Clustal method)

[0378] Genes AtCKX3 and AtCKX5 were not annotated as putative cytokininoxidases in the database and ORFs for these genes were not given.Furthermore, the ORF (and consequently the protein structures) predictedfor AtCKX2 was different from our own prediction and our prediction wasconfirmed by sequencing the AtCKX2 cDNA.

[0379] A comparison of the gene structure of the Arabidopsis AtCKX genes1 to 4 and the maize CKX gene is shown in FIG. 1.

[0380] The predicted proteins encoded by the Arabidopsis AtCKX genesshow between 32% and 41% sequence similarity with the maize protein,while they show between 35% and 66% sequence similarity to each other.Because of this reduced sequence conservation, it is not clear a prioriwhether the Arabidopsis AtCKX genes encode proteins with cytokininoxidase activity. An alignment of the Arabidopsis AtCKX predictedproteins 1 to 4 and the maize CKX gene is shown in FIG. 2.

Example 3 Transgenic Plants Overexpressing AtCKX1 Showed IncreasedCytokinin Oxidase Activity and Altered Plant Morphology

[0381] 1. Description of the Cloning Process

[0382] The following primers were used to PCR amplify the AtCKX1 genefrom Arabidopsis thaliana, accession Columbia (non-homologous sequencesused for cloning are in lower case):

[0383] Sequence of 5′ primer: cggtcgacATGGGATTGACCTCATCCTTACG (SEQ IDNO:13)

[0384] Sequence of 3′ primer: gcgtcgacTTATACAGTTCTAGGTTTCGGCAGTAT (SEQID NO: 14)

[0385] A 2235-bp PCR fragment, amplified by these primers, was insertedin the Sal I site of pUC19. The insert was sequenced and confirmed thatthe PCR amplification product did not contain any mutations. TheSalI/SalI fragment of this vector was subcloned in the SalI sitedownstream of a modified CaMV 35S promoter (carrying three tetracyclineoperator sequences) in the binary vector pBinHyg-Tx (Gatz et al., 1992).The resulting construct was introduced into tobacco and Arabidopsisthaliana through Agrobacterium-mediated transformation, using standardtransformation protocols.

[0386] 2. Molecular Analysis of the Transgenic Lines

[0387] Several transgenic lines were identified that synthesize theAtCKX1 transcript at high levels (FIG. 3). Transgenic lines expressingAtCKX1 transcript also showed increased cytokinin oxidase activity asdetermined by a standard assay for cytokinin oxidase activity based onconversion of [2-³H]iP to adenine as described (Motyka et al., 1996).This is exemplified for 2 tobacco and 2 Arabidopsis lines in Table 6.This result proves that the AtCKX1 gene encodes a protein with cytokininoxidase activity. TABLE 6 Cytokinin oxidase activity in AtCKX1transgenic plant tissues Leaf sample Cytokinin oxidase activity Plantspecies Plant line (nmol Ade/mg protein.h) Arabidopsis Col-0 wild-type0.009 CKX1-11 0.024 CKX1-22 0.026 CKX1-22 0.027 Tobacco SNN wild-type0.004 CKX1-SNN-8 0.016 CKX1-SNN-28 0.021

[0388] 3. Phenotypic Description of the Transgenic Lines

[0389] 3.1 In Tobacco:

[0390] The plants had a dwarfed phenotype with reduced apical dominance(FIG. 7A, B and C) and increased root production (FIG. 8).

[0391] Five Categories of Phenotype:

[0392] 1) strong—2 clones

[0393] 2) intermediate—3 clones

[0394] 3) weak—4 clones

[0395] 4) tall plants (as WT) with large inflorescence—5 clones

[0396] 5) similar to WT, 9 clones

[0397] Height (see FIG. 7B and C)

[0398] WT: between 100-150 cm

[0399] weak: approximately 75 cm

[0400] intermediate: appr. 40-45 cm (main stem app. 25 cm but overgrownby side branches.

[0401] strong: appr. 10 cm

[0402] The transgenics AtCKX148 and AtCKX1-50 displayed a strongphenotype. Below are measurements for stem elongation as compared to WTplants: Line Wild-type AtCKX1-48 AtCKX1-50 Days after germination Height(cm) Height (cm) Height (cm) 47 9.5 ± 0.5 1.3 ± 0.3 1.2 ± 0.2 58 22.4 ±2.3  2.2 ± 0.3 2.3 ± 0.3 68 35.3 ± 2.6  3.1 ± 0.5 2.6 ± 0.5 100 113.3 ±9.8  7.1 ± 0.8 4.8 ± 0.9 117 138.6 ± 8.1  8.7 ± 0.7 6.6 ± 0.9 131 139.0± 9.3  9.3 ± 0.7 8.6 ± 1.0 152 136.6 ± 10.4  10.9 ± 1.1  10.0 ± 1.0  16511.8 ± 1.9  11.4 ± 1.4  181 16.5 ± 1.7  14.9 ± 1.2  198 19.5 ± 1.5  18.1± 1.3 

[0403] Experimental: Plants were grown in soil in a greenhouse. Datawere collected from at least ten plants per line.

[0404] Leaves (see FIGS. 7D and E)

[0405] The shape of leaves of AtCKX1 transgenic expressors waslanceolate (longer and narrow): width-to-length ratio of mature leaveswas reduced from 1:2 in wild type in AtCKX1 transgenics (FIG. 7E). Thenumber of leaves and leaf surface was reduced compared to WT (see FIG.7D). A prominent difference was also noted for progression of leafsenescence. In WT tobacco, leaf senescence starts in the most basalleaves and leads to a uniform reduction of leaf pigment (FIG. 7E). Bycontrast, ageing leaves of strongly expressing AtCKX1 plants stayedgreen along the leaf veins and turned yellow in the intercostal regions,indicating altered leaf senescence. The texture of older leaves was morerigid.

[0406] Roots

[0407] In vitro grown plants highly expressing the gene were easilydistinguishable from the WT by their ability to form more roots whichare thicker (stronger) (FIG. 8A), as well as by forming aerial rootsalong the stem.

[0408] The primary root was longer and the number of lateral andadventitious roots was higher as illustrated in FIG. 8C for AtCKX1-50overexpressing seedlings (see also Example 9).

[0409] The dose-response curve of root growth inhibition by exogenouscytokinin showed that roots of transgenic seedllings are more cytokininresistant than WT roots (FIG. 8D). The resistance of AtCKX1 transgenicsto iPR was less marked than for AtCKX2, which is consistent with thesmaller changes in iP-type cytokinins in the latter (see Table 10).

[0410] A large increase in root biomass was observed for adult plantsgrown in soil (see FIG. 8B for a plant grown in soil for 4 to 5 months)despite the fact that growth of the aerial plant parts was highlyreduced.

[0411] Internode Distance

[0412] intermediate phenotype: the 5^(th) internode below inflorescenceis about 2.5 cm long and 9^(th) internode was about 0.5 cm long comparedto 5 cm and 2 cm for the length of the 5^(th) and 9^(th) internoderespectively, in WT plants.

[0413] strong phenotype: plant AtCKX1-50 The length of the 20^(th)internode from the bottom measured at day 131 after germination was1.3±0.4 mm compared to 39.2±3.8 mm for WT

[0414] Apical Dominance and Branching

[0415] More side branches were formed indicating reduced apicaldominance compared to WT plants during vegetative growth (see FIG. 9).The side branches overgrew the main stem, reaching a height of 40-45 cmfor intermediate AtCKX1 expressors. Even secondary branches appeared.However, the buds were not completely released from apical dominance,i.e. lateral shoots did not really continue to develop. The reducedapical dominance might be due to reduced auxin production by the smallershoot apical meristem (see Example 10).

[0416] Reproductive Development

[0417] The onset of flowering in AtCKX1 transgenics was delayed, thenumber of flowers and the seed yield per capsule was reduced. The sizeof flowers was not altered in transgenic plants and the weight of theindividual seeds was comparable to the weight of seeds from wild typeplants. Data for two representative AtCKX1 transgenics is summarizedbelow: A. Onset of flowering Line Wild-type AtCKX1-48 AtCKX1-50Flowering time 106.2 ± 3.3 193.3 ± 4.3 191.8 ± 3.8 (DAG)

[0418] Experimental: Data collected for at least ten plants per line.The full elongation of the first flower was defined as onset offlowering. DAG=days after germination. B. Number of seed capsules perplant Line Wild-type AtCKX1-48 AtCKX1-50 Number of 83.33 ± 5.13 2.00 ±1.00 2.60 ± 1.67 capsules

[0419] Experimental: Number of seed capsules was determined at leastfrom 5 different plants. Please note that these plants were grown undergreenhouse conditions during winter time. This affects negatively thenumber of flowers that are formed, in particular in the transgenicclones. However, the general picture that they form a reduced number offlowers is correct. n.d., not determined C. Seed yield/capsule (mg) LineWild-type AtCKX1-48 AtCKX1-50 Seed/capsule (mg) 87.41 ± 28.75 23.83 ±13.36 61.8 ± 40.66

[0420] Experimental: Seed yield was determined for at least 12 seedcapsules. The size of seed capsules was very variable, hence the largestandard deviations. n.d., not determined D. Weight of 100 seeds (mg)Line Wild-type AtCKX1-48 AtCKX1-50 Seeds weight (mg) 9.73 ± 0.44 10.70 ±1.60 9.54 ± 0.94

[0421] Experimental: The seed biomass was determined as the weight of100 seed from at least 5 different seed capsules. n.d., not determined

[0422] 3.2 In Arabidopsis

[0423] onset of germination was same as for WT

[0424] the total root system was enlarged and the number of side rootsand adventitious roots was enhanced (see FIGS. 4A through D)

[0425] the growth of aerial organs was reduced resulting in a dwarfedphenotype (see FIGS. 4E and F) and the leaf biomass was reduced. Leafand flower formation is delayed.

[0426] the life cycle was longer compared to VVT and the seed yield waslower compared to WT

[0427] The following morphometric data illustrate these phenotypes:

[0428] Root Development Line Wild-type AtCKX1-11 AtCKX1-15 A. Totallength of the root system Length (mm) 32.5 76.5 68.4 B. Primary rootlength Length (mm) 32.3 ± 3.8 52.3 ± 4.8 39.9 ± 4.2 C. Lateral roots(LR) length Length (mm)  0.2 ± 0.4 15.6 ± 11.0 10.4 ± 7.6 D.Adventitious roots length Length (mm) 0.03 ± 0.18  8.6 ± 8.5 19.1 ± 11.0E. Number of lateral roots (LR) Number of LR  0.3 ± 0.5 10.4 ± 5.4  2.6± 1.1 F. Number of adventitious roots (AR) Number of AR 0.03 ± 0.18  1.6± 1.1  2.6 ± 1.1

[0429] Experimental: Measurements were carried out on plants 8 daysafter germination in vitro on MS medium. At least 17 plants per linewere scored.

[0430] Shoot Development A. Leaf surface AtCKX1-11-7 AtCKX1-11-12AtCKX1-15-1 T3 T3 T3 homozygous homozygous homozygous Line Wild-typeplants plants plants Leaf 21.16 ± 1.73 2.28 ± 0.58 2.62 ± 0.28 1.66 ±0.22 surface (cm²)

[0431] Experimental: Leaf surface area of main rosette leaves formedafter 30 days after germination was measured, 3 plants per clone wereanalysed.

[0432] Reproductive Development

[0433] Onset of flowering AtCKX1-11 AtCKX2-2 AtCKX2-5 T3 T2 T2heterozygous heterozygous heterozygous Line Wild-type plants plantsplants Flowering 43.6 ± 5.8 69.7 ± 9.4 51.2 ± 4.1 45.1 ± 6.9 time (DAG)

[0434] Experimental: Plants were grown under greenhouse condition. Atleast 13 plants per clone were analysed. DAG=days after germination

[0435] Conclusion: The analysis of AtCKX1 transgenic Arabidopsis plantsconfirmed largely the results obtained from tobacco and indicates thegeneral nature of the consequences of a reduced cytokinin content. Thetotal root system was enlarged (the total root length was increased app.110-140% in AtCKX1 transgenics), the shoot developed more slowly(retarded flowering) and the leaf biomass was reduced. The seed yieldwas lower in the transgenics as well (data not shown).

Example 4 Transgenic Plants Overexpressing AtCKX Showed IncreasedCytokinin Oxidase Activity and Altered Plant Morphology

[0436] 1. Description of the Cloning Process

[0437] The following primers were used to PCR amplify the AtCKX2 genefrom Arabidopsis thaliana, accession Columbia (non-homologous sequencesused for cloning are in lower case): Sequence of 5′ primer:gcggtaccAGAGAGAGAAACATAAACAAATGGC (SEQ ID NO:15) Sequence of 3′ primer:gcggtaccCAATTTTACTTCCACCAAAATGC (SEQ ID NO:16)

[0438] A 3104-bp PCR fragment, amplified by these primers, was insertedin the KpnI site of pUC19. The insert was sequenced to check that nodifferences to the published sequence were introduced by the PCRprocedure. The KpnI/KpnI fragment of this vector was subcloned in theKpnI site downstream of a modified CaMV 35S promoter (carrying threetetracycline operator sequences) in the binary vector pBinHyg-Tx (Gatzet al., 1992). The resulting construct was introduced into tobacco andArabidopsis thaliana through Agrobacterium-mediated transformation,using standard transformation protocols.

[0439] 2. Molecular Analysis of the Transgenic Lines

[0440] Several transgenic lines were identified that synthesize theAtCKX2 transcript at high levels (FIG. 6). Transgenic lines expressingAtCKX2 transcript also showed increased cytokinin oxidase activity. Thisis exemplified for 2 tobacco and 3 Arabidopsis lines in Table 7. Thisresult proves that the AtCKX2 gene encodes a protein with cytokininoxidase activity. TABLE 7 Cytokinin oxidase activity in AtCKX2transgenic plant tissues Sample Plant species and Cytokinin oxidaseactivity tissue Plant line (nmol Ade/mg protein.h) Arabidopsis callusCol-0 wild-type 0.037 CKX2-15 0.351 CKX2-17 0.380 CKX2-55 0.265 Tobaccoleaves SNN wild-type 0.009 CKX2-SNN-18 0.091 CKX2-SNN-19 0.091

[0441] 3. Phenotypic Description of the Transgenic Lines

[0442] 3.1 In Tobacco (see FIGS. 7 to 10):

[0443] Three Categories of Phenotype:

[0444] 1) strong—15 clones (similar to intermediate phenotype of AtCKX1)

[0445] 2) weak—6 clones

[0446] 3) others—similar to WT plants, 7 clones

[0447] Aerial Plant Tarts

[0448] The observations concerning plant height, internode distance,branching, leaf form and yellowing were similar as for AtCKX1transgenics with some generally minor quantitative differences in thatthe dwarfing characteristics were more severe in AtCKX1 transgenics thanin AtCKX2 trangenics (compare AtCKX1 plants with AtCKX2 plants in FIGS.7A and B). This is illustrated below for stem elongation and internodedistance measurements of clones with a strong phenotype AtCKX2-38 andAtCKX2-40: Stem elongation Line Wild-type AtCKX2-38 AtCKX2-40 Days afterHeight Height Height germination (cm) (cm) (cm) 47  9.5 ± 0.5  2.4 ± 0.1 2.6 ± 0.2 58  22.4 ± 2.3  5.5 ± 0.7  5.3 ± 0.5 68  35.3 ± 2.6  7.1 ±0.8  7.0 ± 0.7 100 113.3 ± 9.8 15.5 ± 2.5 20.3 ± 6.4 117 138.6 ± 8.119.8 ± 3.8 29.5 ± 6.0 131 139.0 ± 9.3 26.5 ± 7.0 33.4 ± 5.8 152 136.6 ±10.4 33.7 ± 6.3 33.9 ± 6.4 165 36.2 ± 4.3

[0449] Experimental: Plants were grown in soil in a green house. Datawere collected from at least ten plants per line. Internode distanceLine Wild-type AtCKX2-38 Internode distance 39.2 ± 3.8 7.2 ± 1.6 (mm)

[0450] Experimental: The length of the 20^(th) internode from the bottomwas measured at day 131 after germination.

[0451] Roots

[0452] In vitro plants highly expressing the gone were easilydistinguishable from WT plants by ability to form more roots which arethicker (stronger) as well as by forming aerial roots along the stem.

[0453] The primary root was longer and the number of lateral andadventitious roots was higher as illustrated in FIG. 8C for AtCKX2-38overexpressing seedlings (see also Example 9).

[0454] The dose-response curve of root growth inhibition by exogenouscytokinin showed that roots of transgenic seedllings were more cytokininresistant than WT roots (FIG. 8D). The resistance of AtCKX1-28transgenics to iPR was less marked than for AtCKX2-38, which isconsistent with the smaller changes in iP-type cytokinins in the latter(see Table 10).

[0455] An increase in fresh and dry weight of the root biomass of TOlines of AtCKX2 transgenic plants compared to WT was observed for plantgrown in soil, as illustrated in the following table: Line Wild-typeAtCKX2 (T0) Fresh weight 45.2 ± 15.4 77.1 ± 21.3 (g) Dry weight  6.3 ±1.9  8.6 ± 2.2 (g)

[0456] Experimental: Six WT plants and six independent To lines of35S::AtCKX2 clone were grown on soil. After flowering the root systemwas washed with water, the soil was removed as far as possible and thefresh weight and dry weight was measured.

[0457] An increase in fresh and dry weight of the root biomass was alsoobserved for F1 progeny of AtCKX2 transgenics grown in hydroponics ascompared to WT, as illustrated in the following table: Line Wild-typeAtCKX2-38 AtCKX2-40 Fresh weight ROOT 19.76 ± 6.79 33.38 ± 7.76 50.04 ±15.59 (g) Dry weight ROOT  2.36 ± 0.43  2.61 ± 0.39  3.52 ± 1.06 (g)Fresh weight SHOOT 159.8 ± 44.53 33.66 ± 2.67 48.84 ± 11.83 (g) Freshweight  8.24 ± 0.63  1.04 ± 0.18  1.08 ± 0.51 SHOOT/ROOT ratio

[0458] Experimental: Soil grown plants were transferred 60 days aftergermination to a hydroponic system (Hoagland's solution) and grown foradditional 60 days. The hydroponic solution was aerated continuously andreplaced by fresh solution every third day.

[0459] In summary, transgenic plants grown in hydroponic solution formedapproximately 65-150% more root biomass (fresh weight) than wild typeplants. The increase in dry weight was 10-50%. This difference ispossibly in part due to the larger cell volume of the transgenics. Thisreduces the relative portion of cell walls, which forms the bulk of drymatter material. The shoot biomass was reduced to 20%-70% of wild typeshoots. The difference in fresh weight leads to a shift in theshoot/root ratio, which was approximately 8 in wild type butapproximately 1 in the transgenic clones.

[0460] Conclusion:

[0461] An increase in root growth and biomass was observed for AtCKX2transgenic seedlings and adult plants grown under different conditionscompared to WT controls despite the fact that growth of the aerial plantparts is reduced. Quantitative differences were observed betweendifferent transgenic plants: higher increases in root biomass wereobserved for the strongest expressing clones.

[0462] Reproductive Development

[0463] The onset of flowering in AtCKX2 transgenics was delayed, thenumber of flowers and the seed yield per capsule was reduced. Theseeffects were very similar to those observed in the AtCKX1 transgenicplants but they were less prominent in the AtCKX2 transgenics, asindicated in the tables below. The size of flowers was not altered intransgenic plants and the weight of the individual seeds was comparableto the weight of seeds from wild type plants. Onset of flowering AtCKX1-AtCKX1- AtCKX2- AtCKX2- Line Wild-type 48 50 38 40 Flowering 106.2 ± 3.3193.3 ± 191.8 ± 140.6 ± 121.9 ± time 4.3 3.8 6.5 9.8 (DAG)

[0464] Experimental: Data collected for at least ten plants per line.The full elongation of the first flower was defined as onset offlowering. DAG=days after germination. B. Number of seed capsules perplant AtCKX1- AtCKX2- AtCKX2- Line Wild-type AtCKX1-48 50 38 40 Number83.33 ± 5.13 2.00 ± 1.00 2.60 ± 4.30 ± n.d. of 1.67 2.58 capsules

[0465] Experimental: Number of seed capsules was determined at leastfrom 5 different plants. Please note that these plants were grown undergreen house conditions during winter time. This affects negatively thenumber of flowers that are formed, in particular in the transgenicclones. However, the general picture that they form a reduced number offlowers is correct. n.d., not determined C. Seed yield/capsule (mg)AtCKX1- AtCKX1- AtCKX2- AtCKX2- Line Wild-type 48 50 38 40 Seed/ 87.41 ±28.75 23.83 ± 61.8 ± 46.98 ± n.d. capsule 13.36 40.66 29.30 (mg)

[0466] Experimental: Seed yield was determined for at least 12 seedcapsules. The size of seed capsules was very variable, hence the largestandard deviations. n.d., not determined D. Weight of 100 seeds (mg)AtCKX1- AtCKX2- AtCKX2- Line Wild-type AtCKX1-48 50 38 40 Seeds 9.73 ±0.44 10.70 ± 9.54 ± 10.16 ± n.d. weight 1.60 0.94 0.47 (mg)

[0467] Experimental: The seed biomass was determined as the weight of100 seed from at least 5 different seed capsules. n.d., not determined

[0468] 3.2 In Arabidopsis:

[0469] The following morphometric data were obtained for AtCKX2transgenics:

[0470] Root Development Line Wild-type AtCKX2-2 AtCKX2-5 A. Total lengthof the root system Length (mm) 32.5 50.6 48.5 B. Primary root lengthLength (mm) 32.3 ± 3.8 30.7 ± 4.8 31.6 ± 6.8 C. Lateral roots lengthLength (mm)  0.2 ± 0.4  5.5 ± 9.0 1.9 ± 2.5 D. Adventitious roots lengthLength (mm) 0.03 ± 0.18 14.4 ± 10.2 14.9 ± 9.1 E. Number of lateralroots (LR) Number of LR  0.3 ± 0.5  2.9 ± 2.3 1.9 ± 1.0 F. Number ofadventitious roots (AR) Number of AR 0.03 ± 0.18  1.8 ± 0.9 1.8 ± 1.0

[0471] Experimental: Measurements were carried out on plants 8 d.a.g. Invitro on MS medium. At least 17 plants per line were scored.

[0472] Shoot Development Leaf surface AtCKX2-2 AtCKX2-5 AtCKX2-9 T2 T2T2 heterozygous heterozygous heterozygous Line Wild-type plants plantsplants Leaf 21.16 ± 1.73 8.20 ± 2.35 8.22 ± 0.55 7.72 ± 0.85 surface(cm²)

[0473] Experimental: Leaf surface area of main rosette leaves formedafter 30 days after germination was measured. 3 plants per clone wereanalysed.

[0474] Reproductive Development Onset of flowering AtCKX1-11 AtCKX2-2AtCKX2-5 T3 T2 T2 heterozygous heterozygous heterozygous Line Wild-typeplants plants plants Flowering 43.6 ± 5.8 69.7 ± 9.4 51.2 ± 4.1 45.1 ±6.9 time (DAG)

[0475] Experimental: Plants were grown under greenhouse condition. Atleast 13 plants per clone were analysed. DAG=days after germination.

[0476] Conclusion: Arabidopsis AtCKX2 transgenics had reduced leafbiomass and a dwarfing phenotype similar to AtCKX1 transgenics (compareFIG. 5 with FIG. 4F). The total root system was also enlarged in AtCKX2transgenic Arabidopsis. The total root length is increased approximately50% in AtCKX2 transgenics. The AtCKX1 transgenics have longer primaryroots, more side roots and form more adventitious roots. AtCKX2transgenics lack the enhanced growth of the primary root but form moreside roots and lateral roots than W T.

[0477] SUMMARY

[0478] The phenotypes observed for AtCKX2 transgenics were very similarbut not identical to the AtCKX1 transgenics, which in turn were verysimilar but not identical to the results obtained for the tobaccotrangenics. This confirms the general nature of the consequences of areduced cytokinin content in these two plant species and therefore,similar phenotypes can be expected in other plant species as well. Themain difference between tobacco and Arabidopsis is the lack of enhancedprimary root growth in AtCKX2 overexpressing plants (data not shown).

Example 5 Transgenic Plants Overexpressing AtCKX3 Showed IncreasedCytokinin Oxidase Activity and Altered Plant Morphology

[0479] 1. Description of the Cloning Process

[0480] The following primers were used to PCR amplify the AtCKX3 genefrom Arabidopsis thaliana, accession Columbia (non-homologous sequencesused for cloning are in lower case):

[0481] Sequence of 5′ primer gcggtaccTTCATTGATAAGAATCMGCTATTCA (SEQ IDNO:17)

[0482] Sequence of 3′ primer: gcggtaccCAAAGTGGTGAGAACGACTAACA (SEQ IDNO:18)

[0483] A 3397-bp PCR fragment, produced by this PCR amplification, wasinserted in the KpnI site of pBluescript. The insert was sequenced toconfirm that the PCR product has no sequence changes as compared to thegene. The KpnI/KpnI fragment of this vector was subcloned in the KpnIsite downstream of a modified CaMV 35S promoter (carrying threetetracycline operator sequences) in the binary vector pBinHyg-Tx (Gatzet al., 1992). The resulting construct was introduced into tobacco andArabidopsis thaliana through Agrobacterium-mediated transformation,using standard transformation protocols.

[0484] 2. Molecular Analysis of the Transgenic Lines

[0485] Several transgenic tobacco lines were identified that synthesizethe AtCKX3 transcript at high levels (FIG. 11A.). Transgenic tobaccolines expressing AtCKX3 transcript also showed increased cytokininoxidase activity. This is exemplified for three plants in Table 8. Thisproves that the AtCKX3 gene encodes a protein with cytokinin oxidaseactivity. TABLE 8 Cytokinin oxidase activity in AtCKX4 transgenic planttissues Sample Plant species and Cytokinin oxidase activity tissue Plantline (nmol Ade/mg protein.h) tobacco leaves SNN wild-type 0.011CKX3-SNN-3 0.049 CKX3-SNN-6 0.053 CKX3-SNN-21 0.05

[0486] 3. Plant Phenotypic Analysis

[0487] The phenotypes generated by overexpression of the AtCKX3 gene intobacco and Arabidopsis were basically similar as those of AtCKX1 andAtCKX2 expressing plants, i.e. enhanced rooting and dwarfing. However,overexpression of the AtCKX3 gene in tobacco resulted in a strongerphenotype compared to AtCKX2. In this sense AtCKX3 overexpression wasmore similar to AtCKX1 overexpression.

Example 6 Transgenic Plants Overexpressing AtCKX4 Showed IncreasedCytokinin Oxidase Activity and Altered Plant Morphology

[0488] 1. Description of the Cloning Process

[0489] The following primers were used to PCR amplify the AtCKX4 genefrom Arabidopsis thaliana, accession Columbia (non-homologous sequencesused for cloning are in lower case):

[0490] Sequence of 5′ primer: gcggtaccCCCATTAACCTACCCGTTTG (SEQ IDNO:19)

[0491] Sequence of 3′ primer: gcggtaccAGACGATGAACGTACTTGTCTGTA (SEQ IDNO:20)

[0492] A 2890-bp PCR fragment, produced by this PCR amplification, wasinserted in the KpnI site of pBluescript. The Insert was sequenced toconfirm that the PCR product has no sequence changes as compared to thegene. The KpnI/KpnI fragment of this vector was subcloned in the KpnIsite downstream of a modified CaMV 35S promoter (carrying threetetracycline operator sequences) in the binary vector pBinHyg-Tx (Gatzet al., 1992). The resulting construct was introduced into tobacco andArabidopsis thaliana through Agrobacterium-mediated transformation,using standard transformation protocols.

[0493] 2. Molecular Analysis of the Transgenic Lines

[0494] Several transgenic tobacco lines synthesized the AtCKX4transcript at high levels (FIG. 11B.). Transgenic lines expressingAtCKX4 transcript also showed increased cytokinin oxidase activity. Thisis exemplified for 3 Arabidopsis and 3 tobacco lines in Table 9. Thisresult proves that the AtCKX4 gene encodes a protein with cytokininoxidase activity. TABLE 9 Cytokinin oxidase activity in AtCKX4transgenic plant tissues Sample Plant species and Cytokinin oxidaseactivity tissue Plant line (nmol Ade/mg protein.h) Arabidopsis callusCol-0 wild-type 0.037 CKX4-37 0.244 CKX4-40 0.258 CKX4-41 0.320 tobaccoleaves SNN wild-type 0.011 CKX4-SNN-3 0.089 CKX4-SNN-18 0.085CKX4-SNN-27 0.096

[0495] Overall, the data showed that the apparent K_(m) values for thefour cytokinin oxidases were in the range of 0.2 to 9.5 μM with IP assubstrate, which further demonstrates that the proteins encoded byAtCKX1 through 4 are indeed cytokinin oxidase enzymes as disclosedherein.

[0496] 3. Plant Phenotypic Analysis

[0497] The phenotypes generated by overexpression of the AtCKX4 gene intobacco and Arabidopsis were basically similar as those of AtCKX1 andAtCKX2 expressing plants, i.e. enhanced rooting, reduced apicaldominance, dwarfing and yellowing of intercostal regions in older leavesof tobacco. An additional phenotype in tobacco was lanceolate leaves(altered length-to-width ratio).

[0498] General Observations of AtCKX Overexpressing Tobacco Plants

[0499] Overall, the phenotypic analysis demonstrated that AtCKX geneoverexpression caused drastic developmental alterations in the plantshoot and root system in tobacco, including enhanced development of theroot system and dwarfing of the aerial plant part. Other effects such asaltered leaf senescence, formation of adventitious root on stems, andothers were also observed as disclosed herein. The alterations were verysimilar, but not identical, for the different genes. In tobacco, AtCKX1and AtCKX3 overexpressors were alike as were AtCKX2 and AtCKX4.Generally, the two former showed higher expression of the traits,particularly in the shoot. Therefore, a particular cytokinin oxidasegene may be preferred for achieving the phenotypes that are described inthe embodiments of this invention.

Example 7 Cloning of the AtCKX5 Gene

[0500] The following primers were used to PCR amplify the AtCKX5 genefrom Arabidopsis thaliana, accession Columbia (non-homologous sequencesused for cloning are in lower case): Sequence of 5′ primer:ggggtaccTTGATGAATCGTGAAATGAC (SEQ ID NO:21) Sequence of 5′ primer:ggggtaccCTTTCCTCTTGGTTTTGTCCTGT (SEQ ID NO:22)

[0501] The sequence of the 5′ primer includes the two potentialstartcodons of the AtCKX5 protein, the most 5′ startcodon is underlinedand a second ATG is indicated in italics. A 2843-bp PCR fragment,produced by this PCR amplification, was inserted as a blunt-end productin pCR-Blunt II-TOPO cloning vector (Invitrogen).

Example 8 Cloning of the AtCKX6 Gene

[0502] The following primers were used to PCR amplify the AtCKX6 genefrom Arabidopsis thaliana, accession Columbia (non-homologous sequencesused for cloning are in lower case): Sequence of 5′ primer:gctctagaTCAGGAAAAGAACCATGCTTATAG (SEQ ID NO:23) Sequence of 3′ primer:gctctagaTCATGAGTATGAGACTGCCTTTTG (SEQ ID NO:24)

[0503] A 1949-bp PCR fragment, produced by this PCR amplification, wasinserted as a blunt-end product in pCR-Blunt II-TOPO cloning vector(Invitrogen).

Example 9 Tobacco Seedling Growth Test Demonstrated Early Vigor of AtCKXTransgenics

[0504] Seeds of AtCKX1-50 and AtCKX2-38 overexpressing transgenics andWT tobacco were sown in vitro on MS medium, brought to culture room 4days after cold treatment and germinated after 6 days. Observations onseedling growth were made 10 days after germination (see also FIG. 8C)and are summarized below. At least 20 individuals were scored per clone.Similar data have been obtained in two other experiments. Line Wild-typeAtCKX1-50 AtCKX2-38 A. Total length of the root system Length 61.1 122.0106.5 (mm) B. Primary root length Length 32.3 ± 2.6 50.8 ± 4.5 52.4 ±4.8 (mm) C. Lateral roots length Length  9.8 ± 5.5 18.0 ± 8.1 13.0 ± 6.0(mm) D. Adventitious roots length Length 19.0 ± 5.0 53.0 ± 12.0 42.0 ±9.8 (mm) E. Number of lateral roots (LR) Number of LR  1.9 ± 0.9  6.5 ±2.2  5.6 ± 2.0 F. Number of adventitious roots (AR) Number of AR  2.2 ±0.6  3.5 ± 0.9  3.6 ± 1.3

[0505] AtCKX1 and AtCKX2 Plants, General Observations:

[0506] Seedlings of AtCKX1 and AtCKX2 overexpressing tobacco plants had60% more adventitious roots and three times more lateral roots thanuntransformed control plants 10 days after germination. The length ofthe primary root was increased by about 70%. This—together with more andlonger side roots and secondary roots—resulted in a 70-100% increase intotal root length. These results showed that overexpression of cytokininoxidase enhances the growth and development of both the main root andthe adventitious roots, resulting in early vigor.

Example 10 Histological Analysis of Altered Plant Morphology in AtCKX1Overexpressing Tobacco Plants

[0507] Microscopic analysis of different tissues revealed that themorphological changes in AtCKX transgenics are reflected by distinctchanges in cell number and rate of cell formation (see FIG. 10). Theshoot apical meristem (SAM) of AtCKX1 transgenics was smaller than inwild type and fewer cells occupy the space between the central zone andthe peripheral zone of lateral organ fromation, but the cells were ofthe same size (FIG. 10A). The reduced cell number and size of the SAM asa consequence of a reduced cytokinin content indicates that cytokininshave a role in the control of SAM proliferation. No obvious changes inthe differentiation pattern occurred, suggesting that the spatialorganization of the differentiation zones in the SAM is largelyindependent from cell number and from the local cytokinin concentration.The overall tissue pattern of leaves in cytokinin oxidase overexpresserswas unchanged. However, the size of the phloem and xylem wassignificantly reduced (FIG. 10B). By contrast, the average cell size ofleaf parenchyma and epidermal cells was increased four- to fivefold(FIGS. 10C, D). New cells of AtCKX1 transgenics are formed at 3-4% ofthe rate of wild type leaves and final leaf cell number was estimated tobe in the range of 5-6% of wild type. This indicates an absoluterequirement for cytokinins in leaves to maintain the cell divisioncycle. Neither cell size nor cell form of floral organs was altered andseed yield per capsule was similar in wild type and AtCKX transgenicplants. The cell population of root meristems of AtCKX1 transgenicplants was enlarged approximately 4-fold and the cell numbers in boththe central and lateral columnella were enhanced (FIGS. 10E, F). Thefinal root diameter was increased by 60% due to an increased diameter ofall types of root cells. The radial root patterns was Identical in wildtype and transgenics, with the exception that frequently a fourth layerof cortex cells was noted in transgenic roots (FIG. 10G). The increasedcell number and the slightly reduced cell length indicates that theenhanced root growth is due to an increased number of cycling cellsrather than increased cell growth. In the presence of lowered cytokinincontent, root meristem cells must undergo additional rounds of mitosisbefore they leave the meristem and start to elongate. The exit from themeristem is therefore regulated by a mechanism that is sensitive tocytokinins. Apparently, cytokinins have a negative regulatory role inthe root meristem and wild type cytokinin concentrations are inhibitoryto the development of a maximal root system. Therefore, reducing thelevel of active cytokinins by overexpressing cytokinin oxidasesstimulates root development, which results in an increase in the size ofthe root with more lateral and adventitious roots as compared to WTplants.

Example 11 AtCKX1 and AtCKX2-Overexpressing Tobacco Plants had a ReducedCytokinin Content

[0508] Among the 16 different cytokinin metabolites that were measured,the greatest change occurred in the iP-type cytokinins in AtCKX2overexpressers (Table 10): the overall decrease in the content ofiP-type cytokinins is more pronounced in AtCKX2 expressing plants thanin AtCKX1 transgenics. AtCKX1 transgenics showed a stronger phenotype inthe shoot. It is not known which cytokinin metabolite is relevant forthe different traits that were analyzed. It may be that differentcytokinin forms play different roles in the various developmentprocesses. Smaller alterations were noted for Z-type cytokinins, whichcould be due to a different accessibility of the substrate or a lowersubstrate specificity of the protein. The total content of iP and Zmetabolites in individual transgenic clones was between 31% and 63% ofwild type. The cytokinin reserve pool of O-glucosides was also loweredin the transgenics (Table 10). The concentration of N-glucosides andDHZ-type cytokinins was very low and was not or only marginally, alteredin transgenic seedlings (data not shown). TABLE 10 Cytokinin content ofAtCKX transgenic plants. Cytokinin extraction, immunopurification, HPLCseparation and quantification by ELISA methods was carried out asdescribed by Faiss et al., 1997. Three independently pooled samples ofapproximately 100 two week old seedlings (2.5 g per sample) wereanalyzed for each clone. Concentrations are in pmol × g fresh weight⁻¹.Abbreviations: iP, N⁶- (Δ²isopentenyl)adenine; iPR,N⁶-(Δ²isopentenyl)adenine riboside; iPRP, N⁶- (Δ²isopentenyl)adenineriboside 5′-monophosphate; Z, trans-zeatin; ZR, zeatin riboside; ZRP,zeatin riboside 5′-monophosphate; ZOG, zeatin O-glucoside; ZROG, zeatinriboside O-glucoside. Line AtCKX1-2 AtCKX1-28 AtCKX2-38 AtCKX2-40Cytokinin WT % of % of % of % of metabolite Concentration ConcentrationWT Concentration WT Concentration WT Concentration WT IP 5.90 ± 1.804.76 ± 0.82 81 4.94 ± 2.62 84 1.82 ± 0.44 31 2.85 ± 0.62 48 IPR 2.36 ±0.74 1.53 ± 0.14 65 0.75 ± 0.27 32 0.55 ± 0.39 23 0.89 ± 0.07 38 IPRP3.32 ± 0.73 0.87 ± 0.26 26 1.12 ± 0.13 34 0.80 ± 0.48 24 1.68 ± 0.45 51Z 0.24 ± 0.06 0.17 ± 0.02 71 0.22 ± 0.03 92 0.21 ± 0.06 88 0.22 ± 0.0292 ZR 0.60 ± 0.13 0.32 ± 0.12 53 0.34 ± 0.03 57 0.34 ± 0.15 57 0.32 ±0.05 53 ZRP 0.39 ± 0.17 0.42 ± 0.11 107 0.28 ± 0.15 72 0.06 ± 0.01 150.17 ± 0.06 44 ZOG 0.46 ± 0.20 0.32 ± 0.09 70 0.26 ± 0.13 57 0.20 ± 0.0743 0.12 ± 0.02 26 ZROG 0.48 ± 0.17 0.30 ± 0.06 63 0.47 ± 0.02 98 0.23 ±0.05 48 0.30 ± 0.13 63 Total 13.75 8.69 63 8.38 61 4.21 31 6.55 48

Example 12 Grafting Experiments Showed that Dwarfing and Enhanced RootDevelopment due to AtCKX Overexpression is Confined to TransgenicTissues

[0509] To investigate which phenotypic effects of cytokinin oxidaseoverexpression are restricted to expressing tissues, i.e. are cell- ororgan-autonmous traits, grafting experiments were performed. Reciprocalgrafts were made between an AtCKX2 transgenic tobacco plant and a WTtobacco. The transgenic plant used in this experiment was AtCKX2-38,which displayed a strong phenotype characterized by enhanced root growthand reduced development of the aerial plant parts. As described inExample 3 through 6, these were two important phenotypes that resultedfrom cytokinin oxidase overexpression in tobacco and arabidopsis.

[0510] Plants were about 15 cm tall when grafted and the graft junctionwas about 10 cm above the soil. FIG. 12 shows plants 15 weeks aftergrafting. The main results were that: (i) the aerial phenotype of a WTscion grafted on a transgenic rootstock was similar to the WT controlgraft (=WT scion on WT rootstock). Importantly, this showed thatoverexpression of the AtCKX2 transgene in the rootstock did not inducedwarfing of the non-transgenic aerial parts of the plant (see FIG. 12A).Improved root growth of the transgenic rootstock was maintained,indicating that improved root growth of AtCKX transgenics is autonomousand does not depend on an AtCKX transgenic shoot (FIG. 12C).Interestingly, the WT scions grafted on the transgenic rootstocks lookedhealthier and were better developed. Notably, senescence of the basalleaves was retarded in these plants (see FIG. 12A); (ii) the transgenicscion grafted on the WT rootstock looked similar to the aerial part ofthe transgenic plant from which it was derived, i.e. the shoot dwarfingphenotype is also autonomous and not dependent on the improved rootgrowth (see FIG. 12B).

[0511] In addition to the above-mentioned better appearance of WT shootsgrafted on a transgenic rootstock, the formation of adventitious rootson the basal part of WT shoots was noted (FIG. 12D, right plant).Formation of adventitious roots also occurred on the stem of AtCKXtransgenics but not on stems of WT control grafts (FIG. 12D, left plant)and therefore seems to be a non-autonomous trait.

[0512] In summary, it is disclosed in this invention that enhanced rootformation and dwarfing of the shoot in AtCKX overexpressing tobacco areautonomous traits and can be uncoupled by grafting procedures.Surprisingly, grafting of a WT scion on an AtCKX transgenic rootstockresulted in more vigourosly growing plants and retardation of leafsenescence.

[0513] As an alternative to grafting, tissue-specific promoters could beused for uncoupling the autonomous phenotypic effects of cytokininoverexpression. Therefore, it is disclosed in this invention thatcytokinin oxidase overexpression in a tissue specific manner can be usedto alter the morphology of a plant such as the shoot or root system.

Example 13 Expression of an AtCKX Gene Under a Root-Specific Promoter inTransgenic Plants Leads to Increased Root Production

[0514] An AtCKX gene (see example 4) is cloned under control of the rootclavata homolog promoter of Arabidopsis (SEQ ID NO 36), which is apromoter that drives root-specific expression. Other root-specificpromoters may also be used for the purpose of this invention. See Table5 for exemplary root-specific promoters.

[0515] Transgenic plants expressing the AtCKX gene specifically in theroots show increased root production without negatively affecting growthand development of the aerial parts of the plant. Positive effects onleaf senescence and growth of aerial plant parts are observed.

Example 14 Suppression of an AtCKX Gene Under a Senescence-InducedPromoter in Transgenic Plants Leads to Delayed Leaf Senescence andEnhanced Seed Yield

[0516] A chimeric gene construct derived from an AtCKX gene and designedto suppress expression of endogenous cytokinin oxidase gene(s) is clonedunder control of a senescence-induced promoter. For example, promotersderived from senescence-associated genes (SAG) such as the SAG12promoter can be used (Quirino et al., 2000). Transgenic plantssuppressing endogenous cytokinin oxidase gene(s) specifically insenescing leaves show delayed leaf senescence and higher seed yieldwithout negatively affecting the morphology and growth and developmentof the plant.

Example 15 Overexpression of an AtCKX Gene in the Female ReproductiveOrgans Leads to Parthenocarpic Fruit Development

[0517] The open reading frame of an AtCKX gene is cloned under controlof a promoter that confers overexpression in the female reproductiveorgans such as for example the DefH9 promoter from Antirrhinum majus orone of its homologues, which have high expression specificity in theplacenta and ovules. Transgenic plants with enhanced cytokinin oxidaseactivity in these tissues show parthenocarpic fruit development.

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[0573]

1 36 1 2236 DNA Arabidopsis thaliana 1 atgggattga cctcatcctt acggttccatagacaaaaca acaagacttt cctcggaatc 60 ttcatgatct tagttctaag ctgtataccaggtagaacca atctttgttc caatcattct 120 gttagtaccc caaaagaatt accttcttcaaatccttcag atattcgttc ctcattagtt 180 tcactagatt tggagggtta tataagcttcgacgatgtcc acaatgtggc caaggacttt 240 ggcaacagat accagttacc acctttggcaattctacatc caaggtcagt ttttgatatt 300 tcatcgatga tgaagcatat agtacatctgggctccacct caaatcttac agtagcagct 360 agaggccatg gtcactcgct tcaaggacaagctctagctc atcaaggtgt tgtcatcaaa 420 atggagtcac ttcgaagtcc tgatatcaggatttataagg ggaagcaacc atatgttgat 480 gtctcaggtg gtgaaatatg gataaacattctacgcgaga ctctaaaata cggtctttca 540 ccaaagtcct ggacagacta ccttcatttgaccgttggag gtacactatc taatgctgga 600 atcagcggtc aagcattcaa gcatggaccccaaatcaaca acgtctacca gctagagatt 660 gttacaggta tttcattcat gctttatctctgcggtagtc tcaaaaaaat atgcacctgt 720 aaagaatatc catctcttca tgagcaaaaacactgacgac tttaaataat ttttgactat 780 aaaacaagag tgcataggca caaatgtgaaatatgcaaca cacaattgta acttgcacca 840 agaaaaaagt tataaaaaca aacaactgataagcaatata tttccaatat ttaatcaggg 900 aaaggagaag tcgtaacctg ttctgagaagcggaattctg aacttttctt cagtgttctt 960 ggcgggcttg gacagtttgg cataatcacccgggcacgga tctctcttga accagcaccg 1020 catatggtaa agttctatct tgaacaaagttcaaacaata tacgctatga ttctaagaac 1080 cactttcctg acacagtcaa ataacttttaataggttaaa tggatcaggg tactctactc 1140 tgacttttct gcattttcaa gggaccaagaatatctgatt tcgaaggaga aaacttttga 1200 ttacgttgaa ggatttgtga taatcaatagaacagacctt ctcaataatt ggcgatcgtc 1260 attcagtccc aacgattcca cacaggcaagcagattcaag tcagatggga aaactcttta 1320 ttgcctagaa gtggtcaaat atttcaacccagaagaagct agctctatgg atcaggtaag 1380 atgtgaaagc aatatataac tagacttagtttccacagag agctccaaat caaccgttgg 1440 ctactagcct actaacataa tgaatggttgccgtgcagga aactggcaag ttactttcag 1500 agttaaatta tattccatcc actttgttttcatctgaagt gccatatatc gagtttctgg 1560 atcgcgtgca tatcgcagag agaaaactaagagcaaaggg tttatgggag gttccacatc 1620 cctggctgaa tctcctgatt cctaagagcagcatatacca atttgctaca gaagttttca 1680 acaacattct cacaagcaac aacaacggtcctatccttat ttatccagtc aatcaatcca 1740 agtaagtgag caaaatgcca aaagcaaatgcgtccagtga ttctgaaaca taaattacta 1800 accatatcca acattttgtg gtttcaggtggaagaaacat acatctttga taactccaaa 1860 tgaagatata ttctatctcg tagcctttctcccctctgca gtgccaaatt cctcagggaa 1920 aaacgatcta gagtaccttt tgaaacaaaaccaaagagtt atgaacttct gcgcagcagc 1980 aaacctcaac gtgaagcagt atttgccccattatgaaact caaaaagagt ggaaatcaca 2040 ctttggcaaa agatgggaaa catttgcacagaggaaacaa gcctacgacc ctctagcgat 2100 tctagcacct ggccaaagaa tattccaaaagacaacagga aaattatctc ccatccaact 2160 cgcaaagtca aaggcaacag gaagtcctcaaaggtaccat tacgcatcaa tactgccgaa 2220 acctagaact gtataa 2236 2 575 PRTArabidopsis thaliana 2 Met Gly Leu Thr Ser Ser Leu Arg Phe His Arg GlnAsn Asn Lys Thr 1 5 10 15 Phe Leu Gly Ile Phe Met Ile Leu Val Leu SerCys Ile Pro Gly Arg 20 25 30 Thr Asn Leu Cys Ser Asn His Ser Val Ser ThrPro Lys Glu Leu Pro 35 40 45 Ser Ser Asn Pro Ser Asp Ile Arg Ser Ser LeuVal Ser Leu Asp Leu 50 55 60 Glu Gly Tyr Ile Ser Phe Asp Asp Val His AsnVal Ala Lys Asp Phe 65 70 75 80 Gly Asn Arg Tyr Gln Leu Pro Pro Leu AlaIle Leu His Pro Arg Ser 85 90 95 Val Phe Asp Ile Ser Ser Met Met Lys HisIle Val His Leu Gly Ser 100 105 110 Thr Ser Asn Leu Thr Val Ala Ala ArgGly His Gly His Ser Leu Gln 115 120 125 Gly Gln Ala Leu Ala His Gln GlyVal Val Ile Lys Met Glu Ser Leu 130 135 140 Arg Ser Pro Asp Ile Arg IleTyr Lys Gly Lys Gln Pro Tyr Val Asp 145 150 155 160 Val Ser Gly Gly GluIle Trp Ile Asn Ile Leu Arg Glu Thr Leu Lys 165 170 175 Tyr Gly Leu SerPro Lys Ser Trp Thr Asp Tyr Leu His Leu Thr Val 180 185 190 Gly Gly ThrLeu Ser Asn Ala Gly Ile Ser Gly Gln Ala Phe Lys His 195 200 205 Gly ProGln Ile Asn Asn Val Tyr Gln Leu Glu Ile Val Thr Gly Lys 210 215 220 GlyGlu Val Val Thr Cys Ser Glu Lys Arg Asn Ser Glu Leu Phe Phe 225 230 235240 Ser Val Leu Gly Gly Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala Arg 245250 255 Ile Ser Leu Glu Pro Ala Pro His Met Val Lys Trp Ile Arg Val Leu260 265 270 Tyr Ser Asp Phe Ser Ala Phe Ser Arg Asp Gln Glu Tyr Leu IleSer 275 280 285 Lys Glu Lys Thr Phe Asp Tyr Val Glu Gly Phe Val Ile IleAsn Arg 290 295 300 Thr Asp Leu Leu Asn Asn Trp Arg Ser Ser Phe Ser ProAsn Asp Ser 305 310 315 320 Thr Gln Ala Ser Arg Phe Lys Ser Asp Gly LysThr Leu Tyr Cys Leu 325 330 335 Glu Val Val Lys Tyr Phe Asn Pro Glu GluAla Ser Ser Met Asp Gln 340 345 350 Glu Thr Gly Lys Leu Leu Ser Glu LeuAsn Tyr Ile Pro Ser Thr Leu 355 360 365 Phe Ser Ser Glu Val Pro Tyr IleGlu Phe Leu Asp Arg Val His Ile 370 375 380 Ala Glu Arg Lys Leu Arg AlaLys Gly Leu Trp Glu Val Pro His Pro 385 390 395 400 Trp Leu Asn Leu LeuIle Pro Lys Ser Ser Ile Tyr Gln Phe Ala Thr 405 410 415 Glu Val Phe AsnAsn Ile Leu Thr Ser Asn Asn Asn Gly Pro Ile Leu 420 425 430 Ile Tyr ProVal Asn Gln Ser Lys Trp Lys Lys His Thr Ser Leu Ile 435 440 445 Thr ProAsn Glu Asp Ile Phe Tyr Leu Val Ala Phe Leu Pro Ser Ala 450 455 460 ValPro Asn Ser Ser Gly Lys Asn Asp Leu Glu Tyr Leu Leu Lys Gln 465 470 475480 Asn Gln Arg Val Met Asn Phe Cys Ala Ala Ala Asn Leu Asn Val Lys 485490 495 Gln Tyr Leu Pro His Tyr Glu Thr Gln Lys Glu Trp Lys Ser His Phe500 505 510 Gly Lys Arg Trp Glu Thr Phe Ala Gln Arg Lys Gln Ala Tyr AspPro 515 520 525 Leu Ala Ile Leu Ala Pro Gly Gln Arg Ile Phe Gln Lys ThrThr Gly 530 535 540 Lys Leu Ser Pro Ile Gln Leu Ala Lys Ser Lys Ala ThrGly Ser Pro 545 550 555 560 Gln Arg Tyr His Tyr Ala Ser Ile Leu Pro LysPro Arg Thr Val 565 570 575 3 2991 DNA Arabidopsis thaliana 3 atggctaatcttcgtttaat gatcacttta atcacggttt taatgatcac caaatcatca 60 aacggtattaaaattgattt acctaaatcc cttaacctca ccctctctac cgatccttcc 120 atcatctccgcagcctctca tgacttcgga aacataacca ccgtgacccc cggcggcgta 180 atctgcccctcctccaccgc tgatatctct cgtctcctcc aatacgccgc aaacggaaaa 240 agtacattccaagtagcggc tcgtggccaa ggccactcct taaacggcca agcctcggtc 300 tccggcggagtaatcgtcaa catgacgtgt atcactgacg tggtggtttc aaaagacaag 360 aagtacgctgacgtggcggc cgggacgtta tgggtggatg tgcttaagaa gacggcggag 420 aaaggggtgtcgccggtttc ttggacggat tatttgcata taaccgtcgg aggaacgttg 480 tcgaatggtggaattggtgg tcaagtgttt cgaaacggtc ctcttgttag taacgtcctt 540 gaattggacgttattactgg tacgcatctt ctaaactttg atgtacatac aacaacaaaa 600 actgtttttgttttatagta tttttcattt tttgtaccat aggttttatg ttttatagtt 660 gtgctaaacttcttgcacca cacgtaagtc ttcgaaacac aaaatgcgta acgcatctat 720 atgttttttgtacatattga atgttgttca tgagaaataa agtaattaca tatacacaca 780 tttattgtcgtacatatata aataattaaa gacaaatttt cacaattggt agcgtgttaa 840 tttgggatttttgtaatgta catgcatgac gcatgcatat ggagcttttc ggttttctta 900 gatttgtgtagtatttcaaa tatatcattt attttctttc gaataaagag gtggtatatt 960 tttaaaatagcaacatttca gaatttttct ttgaatttac actttttaaa ttgttattgt 1020 taatatggattttgaataaa taatttcagg gaaaggtgaa atgttgacat gctcgcgaca 1080 gctaaacccagaattgttct atggagtgtt aggaggtttg ggtcaatttg gaattataac 1140 gagagccagaattgttttgg accatgcacc taaacgggta cgtatcatca tattttacca 1200 tttgttttagtcagcattca tttttcatta gtaattccgt ttcaatttct aaattttttt 1260 agtcaatagaaaatgattct tatgtcagag cttgattatt tagtgatttt tattgagata 1320 aaataaaatataacctaacg gaaataatta ttttactaat cggataatgt ctgattaaaa 1380 cattttatgatattacacta agagagttag agacgtatgg atcacaaaac atgaagcttt 1440 cttagatggtatcctaaaac taaagttagg tacaagtttg gaatttaggt caaatgctta 1500 agttgcattaatttgaacaa aatctatgca ttgaataaaa aaaagatatg gattatttta 1560 taaagtatagtccttgtaat cctaggactt gttgtctaat cttgtcttat gcgtgcaaat 1620 ctttttgatgtcaatatata atccttgttt attagagtca agctctttca ttagtcaact 1680 actcaaatatactccaaagt ttagaatata gtcttctgac taattagaat cttacaaccg 1740 ataaacgttacaatttggtt atcattttaa aaaacagatt tggtcataat atacgatgac 1800 gttctgttttagtttcatct attcacaaat tttatataat tattttcaag aaaatattga 1860 aatactatactgtaatatgg tttctttata tatgtgtgta taaattaaat gggattgttt 1920 tctctaaatgaaattgtgta ggccaaatgg tttcggatgc tctacagtga tttcacaact 1980 tttacaaaggaccaagaacg tttgatatca atggcaaacg atattggagt cgactattta 2040 gaaggtcaaatatttctatc aaacggtgtc gttgacacct cttttttccc accttcagat 2100 caatctaaagtcgctgatct agtcaagcaa cacggtatca tctatgttct tgaagtagcc 2160 aagtattatgatgatcccaa tctccccatc atcagcaagg tactacacat ttacattttc 2220 atcatcgtttttatcatacc ataagatatt taaatgattc atcattgcac cacattaaga 2280 tattcatcatcatcatcgtt acattttttt ttgcatctta tgcttctcat aatctactat 2340 tgtgtaggttattgacacat taacgaaaac attaagttac ttgcccgggt tcatatcaat 2400 gcacgacgtggcctacttcg atttcttgaa ccgtgtacat gtcgaagaaa ataaactcag 2460 atctttgggattatgggaac ttcctcatcc ttggcttaac ctctacgttc ctaaatctcg 2520 gattctcgattttcataacg gtgttgtcaa agacattctt cttaagcaaa aatcagcttc 2580 gggactcgctcttctctatc caacaaaccg gaataagtac atacttctct tcattcatat 2640 ttatcttcaagaaccaaagt aaataaattt ctatgaactg attatgctgt tattgttaga 2700 tgggacaatcgtatgtcggc gatgatacca gagatcgatg aagatgttat atatattatc 2760 ggactactacaatccgctac cccaaaggat cttccagaag tggagagcgt taacgagaag 2820 ataattaggttttgcaagga ttcaggtatt aagattaagc aatatctaat gcattatact 2880 agtaaagaagattggattga gcattttgga tcaaaatggg atgatttttc gaagaggaaa 2940 gatctatttgatcccaagaa actgttatct ccagggcaag acatcttttg a 2991 4 501 PRT Arabidopsisthaliana 4 Met Ala Asn Leu Arg Leu Met Ile Thr Leu Ile Thr Val Leu MetIle 1 5 10 15 Thr Lys Ser Ser Asn Gly Ile Lys Ile Asp Leu Pro Lys SerLeu Asn 20 25 30 Leu Thr Leu Ser Thr Asp Pro Ser Ile Ile Ser Ala Ala SerHis Asp 35 40 45 Phe Gly Asn Ile Thr Thr Val Thr Pro Gly Gly Val Ile CysPro Ser 50 55 60 Ser Thr Ala Asp Ile Ser Arg Leu Leu Gln Tyr Ala Ala AsnGly Lys 65 70 75 80 Ser Thr Phe Gln Val Ala Ala Arg Gly Gln Gly His SerLeu Asn Gly 85 90 95 Gln Ala Ser Val Ser Gly Gly Val Ile Val Asn Met ThrCys Ile Thr 100 105 110 Asp Val Val Val Ser Lys Asp Lys Lys Tyr Ala AspVal Ala Ala Gly 115 120 125 Thr Leu Trp Val Asp Val Leu Lys Lys Thr AlaGlu Lys Gly Val Ser 130 135 140 Pro Val Ser Trp Thr Asp Tyr Leu His IleThr Val Gly Gly Thr Leu 145 150 155 160 Ser Asn Gly Gly Ile Gly Gly GlnVal Phe Arg Asn Gly Pro Leu Val 165 170 175 Ser Asn Val Leu Glu Leu AspVal Ile Thr Gly Lys Gly Glu Met Leu 180 185 190 Thr Cys Ser Arg Gln LeuAsn Pro Glu Leu Phe Tyr Gly Val Leu Gly 195 200 205 Gly Leu Gly Gln PheGly Ile Ile Thr Arg Ala Arg Ile Val Leu Asp 210 215 220 His Ala Pro LysArg Ala Lys Trp Phe Arg Met Leu Tyr Ser Asp Phe 225 230 235 240 Thr ThrPhe Thr Lys Asp Gln Glu Arg Leu Ile Ser Met Ala Asn Asp 245 250 255 IleGly Val Asp Tyr Leu Glu Gly Gln Ile Phe Leu Ser Asn Gly Val 260 265 270Val Asp Thr Ser Phe Phe Pro Pro Ser Asp Gln Ser Lys Val Ala Asp 275 280285 Leu Val Lys Gln His Gly Ile Ile Tyr Val Leu Glu Val Ala Lys Tyr 290295 300 Tyr Asp Asp Pro Asn Leu Pro Ile Ile Ser Lys Val Ile Asp Thr Leu305 310 315 320 Thr Lys Thr Leu Ser Tyr Leu Pro Gly Phe Ile Ser Met HisAsp Val 325 330 335 Ala Tyr Phe Asp Phe Leu Asn Arg Val His Val Glu GluAsn Lys Leu 340 345 350 Arg Ser Leu Gly Leu Trp Glu Leu Pro His Pro TrpLeu Asn Leu Tyr 355 360 365 Val Pro Lys Ser Arg Ile Leu Asp Phe His AsnGly Val Val Lys Asp 370 375 380 Ile Leu Leu Lys Gln Lys Ser Ala Ser GlyLeu Ala Leu Leu Tyr Pro 385 390 395 400 Thr Asn Arg Asn Lys Trp Asp AsnArg Met Ser Ala Met Ile Pro Glu 405 410 415 Ile Asp Glu Asp Val Ile TyrIle Ile Gly Leu Leu Gln Ser Ala Thr 420 425 430 Pro Lys Asp Leu Pro GluVal Glu Ser Val Asn Glu Lys Ile Ile Arg 435 440 445 Phe Cys Lys Asp SerGly Ile Lys Ile Lys Gln Tyr Leu Met His Tyr 450 455 460 Thr Ser Lys GluAsp Trp Ile Glu His Phe Gly Ser Lys Trp Asp Asp 465 470 475 480 Phe SerLys Arg Lys Asp Leu Phe Asp Pro Lys Lys Leu Leu Ser Pro 485 490 495 GlyGln Asp Ile Phe 500 5 3302 DNA Arabidopsis thaliana 5 atggcgagttataatcttcg ttcacaagtt cgtcttatag caataacaat agtaatcatc 60 attactctctcaactccgat cacaaccaac acatcaccac aaccatggaa tatcctttca 120 cacaacgaattcgccggaaa actcacctcc tcctcctcct ccgtcgaatc agccgccaca 180 gatttcggccacgtcaccaa aatcttccct tccgccgtct taatcccttc ctccgttgaa 240 gacatcacagatctcataaa actctctttt gactctcaac tgtcttttcc tttagccgct 300 cgtggtcacggacacagcca ccgtggccaa gcctcggcta aagacggagt tgtggtcaac 360 atgcggtccatggtaaaccg ggatcgaggt atcaaggtgt ctaggacctg tttatatgtt 420 gacgtggacgctgcgtggct atggattgag gtgttgaata aaactttgga gttagggtta 480 acgccggtttcttggacgga ttatttgtat ttaacagtcg gtgggacgtt atcaaacggc 540 ggaattagtggacaaacgtt tcggtacggt ccacagatca ctaatgttct agagatggat 600 gttattactggtacgtacca cgatcttttt cacacagaga ttaaaaaaaa cagtaatagt 660 gattttaacttcgtacgttt ctgatagaca acaaagaact tcgtacgttt ttcgaagttt 720 tttcgtctttttcattttag atctgcgcgg ccatttttgg ttatgctatt gtttgtttgt 780 attgtttgtctctgtttatt tatttctcga acttgttgat agcttttctt cttttcacac 840 atcaatctaatcaccttttt tggtcttaag attagaaaga agatacggac taggtaaaaa 900 taggtggttgtaaacgtaga cgcattaaaa aaatattggt ttttttattt tttgataagc 960 aaaattggtggttggtctaa gattataaac ttgatattaa tgcaaaggtc gatctagcaa 1020 tagaagattaatcaatattc ttggtgtttt aacaacagat tatttcatca ttaaaatcgt 1080 gaaacaaagaaattttggta gtatacatta cgtgtagttt tgttagttta ttaaaaaaaa 1140 tagtatatagttttgttaaa acgcgattta tttagtaaca cattagtata ttacacgttt 1200 aaccaactaaactttttttt ttgaataatt atgttctata tttcttactc aaattatgca 1260 aatttcgtggattcgaagtc aaatttctgc gaaatttaca tggtcatata ttataaaact 1320 gttcatataacccggtgaac aaacagacaa ttaagggttt gaatggttac ggcggttggg 1380 gcggacacaaccgtcaatag atcagaccgt tttttattta ccattcatca attatattcc 1440 gcagtggtttggggtaaaaa aaatagaaga aaaccgcagc ggaccaattc cataccgttt 1500 ttacatacaaataaacatgg tgcgcaacgg tttattgtcc gcctcaaaaa tgaaatggac 1560 taaaccgcagataaattaga ccgctttgtc cgctgcctcc attcatagac taaaaaaaaa 1620 caaccaaaaaaaaaatggtc ccacgcccat gattttacac gaggtttctt gtggcgtaag 1680 gacaaaactcaaaagttcat aacgtttggt cctaaccagg tgtaatggat taagtaacag 1740 tcaattttcttattatagct gtatccatta tgtccacata tgcatccata tacattacac 1800 tgttggtctcaagtgtagtt agattacgaa gactttcaag ttccattttt tggttaggag 1860 ataaacataatttaatgata ccgactttag cactctaggc tcaaaacaag tacagaagag 1920 aatagttttatttcaaactc gttgcattgt tgtatcaatt aattgtgtta gtctttgtat 1980 attcttacataacggtccaa gtttgttgaa atagtttact tactaaactt ttcctaatgg 2040 ggtcaaattttattttatag gaaaaggaga gattgcaact tgttccaagg acatgaactc 2100 ggatcttttcttcgcggtgt taggaggttt gggtcaattc ggcattataa caagagccag 2160 aattaaacttgaagtagctc cgaaaagggt atgttaaatt tgtaaattat gcaactacag 2220 aaaattctatgaaatttatg aatgaacata tatgcatttt tggatttttg taggccaagt 2280 ggttaaggtttctatacata gatttctccg aattcacaag agatcaagaa cgagtgatat 2340 cgaaaacggacggtgtagat ttcttagaag gttccattat ggtggaccat ggcccaccgg 2400 ataactggagatccacgtat tatccaccgt ccgatcactt gaggatcgcc tcaatggtca 2460 aacgacatcgtgtcatctac tgccttgaag tcgtcaagta ttacgacgaa acttctcaat 2520 acacagtcaacgaggtccgt acatacatac aatcataaat catacatgta taattgggag 2580 atctttatgcattattcaat tatattaatt tactttagtt atttaactta tgcaggaaat 2640 ggaggagttaagcgatagtt taaaccatgt aagagggttt atgtacgaga aagatgtgac 2700 gtatatggatttcctaaacc gagttcgaac cggagagcta aacctgaaat ccaaaggcca 2760 atgggatgttccacatccat ggcttaatct cttcgtacca aaaactcaaa tctccaaatt 2820 tgatgatggtgtttttaagg gtattatcct aagaaataac atcactagcg gtcctgttct 2880 tgtttatcctatgaatcgca acaagtaagt ttaactcgat attgcaaaat ttactatcta 2940 cattttcgttttggaatccg aaatattctt acaagctaat tttatgcggc gtttttaggt 3000 ggaatgatcggatgtctgcc gctatacccg aggaagatgt attttatgcg gtagggtttt 3060 taagatccgcgggttttgac aattgggagg cttttgatca agaaaacatg gaaatactga 3120 agttttgtgaggatgctaat atgggggtta tacaatatct tccttatcat tcatcacaag 3180 aaggatgggttagacatttt ggtccgaggt ggaatatttt cgtagagaga aaatataaat 3240 atgatcccaaaatgatatta tcaccgggac aaaatatatt tcaaaaaata aactcgagtt 3300 ag 3302 6523 PRT Arabidopsis thaliana 6 Met Ala Ser Tyr Asn Leu Arg Ser Gln ValArg Leu Ile Ala Ile Thr 1 5 10 15 Ile Val Ile Ile Ile Thr Leu Ser ThrPro Ile Thr Thr Asn Thr Ser 20 25 30 Pro Gln Pro Trp Asn Ile Leu Ser HisAsn Glu Phe Ala Gly Lys Leu 35 40 45 Thr Ser Ser Ser Ser Ser Val Glu SerAla Ala Thr Asp Phe Gly His 50 55 60 Val Thr Lys Ile Phe Pro Ser Ala ValLeu Ile Pro Ser Ser Val Glu 65 70 75 80 Asp Ile Thr Asp Leu Ile Lys LeuSer Phe Asp Ser Gln Leu Ser Phe 85 90 95 Pro Leu Ala Ala Arg Gly His GlyHis Ser His Arg Gly Gln Ala Ser 100 105 110 Ala Lys Asp Gly Val Val ValAsn Met Arg Ser Met Val Asn Arg Asp 115 120 125 Arg Gly Ile Lys Val SerArg Thr Cys Leu Tyr Val Asp Val Asp Ala 130 135 140 Ala Trp Leu Trp IleGlu Val Leu Asn Lys Thr Leu Glu Leu Gly Leu 145 150 155 160 Thr Pro ValSer Trp Thr Asp Tyr Leu Tyr Leu Thr Val Gly Gly Thr 165 170 175 Leu SerAsn Gly Gly Ile Ser Gly Gln Thr Phe Arg Tyr Gly Pro Gln 180 185 190 IleThr Asn Val Leu Glu Met Asp Val Ile Thr Gly Lys Gly Glu Ile 195 200 205Ala Thr Cys Ser Lys Asp Met Asn Ser Asp Leu Phe Phe Ala Val Leu 210 215220 Gly Gly Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala Arg Ile Lys Leu 225230 235 240 Glu Val Ala Pro Lys Arg Ala Lys Trp Leu Arg Phe Leu Tyr IleAsp 245 250 255 Phe Ser Glu Phe Thr Arg Asp Gln Glu Arg Val Ile Ser LysThr Asp 260 265 270 Gly Val Asp Phe Leu Glu Gly Ser Ile Met Val Asp HisGly Pro Pro 275 280 285 Asp Asn Trp Arg Ser Thr Tyr Tyr Pro Pro Ser AspHis Leu Arg Ile 290 295 300 Ala Ser Met Val Lys Arg His Arg Val Ile TyrCys Leu Glu Val Val 305 310 315 320 Lys Tyr Tyr Asp Glu Thr Ser Gln TyrThr Val Asn Glu Glu Met Glu 325 330 335 Glu Leu Ser Asp Ser Leu Asn HisVal Arg Gly Phe Met Tyr Glu Lys 340 345 350 Asp Val Thr Tyr Met Asp PheLeu Asn Arg Val Arg Thr Gly Glu Leu 355 360 365 Asn Leu Lys Ser Lys GlyGln Trp Asp Val Pro His Pro Trp Leu Asn 370 375 380 Leu Phe Val Pro LysThr Gln Ile Ser Lys Phe Asp Asp Gly Val Phe 385 390 395 400 Lys Gly IleIle Leu Arg Asn Asn Ile Thr Ser Gly Pro Val Leu Val 405 410 415 Tyr ProMet Asn Arg Asn Lys Trp Asn Asp Arg Met Ser Ala Ala Ile 420 425 430 ProGlu Glu Asp Val Phe Tyr Ala Val Gly Phe Leu Arg Ser Ala Gly 435 440 445Phe Asp Asn Trp Glu Ala Phe Asp Gln Glu Asn Met Glu Ile Leu Lys 450 455460 Phe Cys Glu Asp Ala Asn Met Gly Val Ile Gln Tyr Leu Pro Tyr His 465470 475 480 Ser Ser Gln Glu Gly Trp Val Arg His Phe Gly Pro Arg Trp AsnIle 485 490 495 Phe Val Glu Arg Lys Tyr Lys Tyr Asp Pro Lys Met Ile LeuSer Pro 500 505 510 Gly Gln Asn Ile Phe Gln Lys Ile Asn Ser Ser 515 5207 2782 DNA Arabidopsis thaliana 7 atgactaata ctctctgttt aagcctcatcaccctaataa cgctttttat aagtttaacc 60 ccaaccttaa tcaaatcaga tgagggcattgatgttttct tacccatatc actcaacctt 120 acggtcctaa ccgatccctt ctccatctctgccgcttctc acgacttcgg taacataacc 180 gacgaaaatc ccggcgccgt cctctgcccttcctccacca cggaggtggc tcgtctcctc 240 cgtttcgcta acggaggatt ctcttacaataaaggctcaa ccagccccgc gtctactttc 300 aaagtggctg ctcgaggcca aggccactccctccgtggcc aagcctctgc acccggaggt 360 gtcgtcgtga acatgacgtg tctcgccatggcggctaaac cagcggcggt tgttatctcg 420 gcagacggga cttacgctga cgtggctgccgggacgatgt gggtggatgt tctgaaggcg 480 gcggtggata gaggcgtctc gccggttacatggacggatt atttgtatct cagcgtcggc 540 gggacgttgt cgaacgctgg aatcggtggtcagacgttta gacacggccc tcagattagt 600 aacgttcatg agcttgacgt tattaccggtacgtaaatac caaaacttca ctaatctcgt 660 tacaattttt taattttttg gtaatataaattttgtacgg ctcaactctt aattaagaat 720 gaaacagtat ctatgatctt ctagatgctctttttttgtc tgcaagcttt aattgtagta 780 acatcagcga tatatatatc acatgcatgtgtattattga tgataatata taatgtttta 840 gttacaaatt tgattctcaa ggtaaaactcacacgccata accagtataa aactccaaaa 900 atcacgtttt ggtcagaaat acatatccttcattaacagt agttatgcta taatttgtga 960 ttataaataa ctccggagtt tgttcacaatactaaatttc aggaaaaggt gaaatgatga 1020 cttgctctcc aaagttaaac cctgaattgttctatggagt tttaggaggt ttgggtcaat 1080 tcggtattat aacgagggcc aggattgcgttggatcatgc acccacaagg gtatgtatca 1140 tgcatctata gtgtaatcaa tttataattttaatgtagtg gtcctaaatc caaaatttga 1200 tttgatttgg ttggaacgta cgtatatataataagtcaaa aggctgattt tgaagacgaa 1260 tttatatact tttgttgaat taaatctgattttgcttacg ttttattaga ttctgcgtaa 1320 taaatcctag gacttgctcg agtgtaatcttgtcttatgc ttgcaaatct tgttgatgtc 1380 aatatctaat cttttttatt atatttccctacgtaagttt tagatatagt tattttaaac 1440 tgctataaat tgtgtacgta tagactttagataaaaagtt gtggtcgctt gcacctattt 1500 gtttatcgct atagtgattc aaaggtctatatatgattct tggtttttct ttttgaaaaa 1560 aatagaccat acaatccaag gaagatgatcttaaatggac taatttatgg atataaattg 1620 atatacaaat ctgcaggtga aatggtctcgcatactctac agtgacttct cggcttttaa 1680 aagagaccaa gagcgtttaa tatcaatgaccaatgatctc ggagttgact ttttggaagg 1740 tcaacttatg atgtcaaatg gcttcgtagacacctctttc ttcccactct ccgatcaaac 1800 aagagtcgca tctcttgtga atgaccaccggatcatctat gttctcgaag tagccaagta 1860 ttatgacaga accacccttc ccattattgaccaggtacta aaatccatta ttcatgatga 1920 ttatcttcac acaatcagta tcatcaccaattaccatcat cacttgtcat atatgatcca 1980 aagtaaatat atcacatgat ataaataaatcgttcaaatc ttttttttta aagaataaaa 2040 gaatcatttt caagcattac tcatacacatctacgaatca ccgtgaccat atataaccat 2100 acgcttatta aataatcatt tttgtttgtaggtgattgac acgttaagta gaactctagg 2160 tttcgctcca gggtttatgt tcgtacaagatgttccgtat ttcgatttct tgaaccgtgt 2220 ccgaaacgaa gaagataaac tcagatctttaggactatgg gaagttcctc atccatggct 2280 taacatcttt gtcccggggt ctcgaatccaagattttcat gatggtgtta ttaatggcct 2340 tcttctaaac caaacctcaa cttctggtgttactctcttc tatcccacaa accgaaacaa 2400 gtaaatattt actttttgat tttgttttatttgaaagtat atcccaataa tgtatgttaa 2460 attgttaaca agaatttatt ttattaatagatggaacaac cgcatgtcaa cgatgacacc 2520 ggacgaagat gttttttatg tgatcggattactgcaatca gctggtggat ctcaaaattg 2580 gcaagaactt gaaaatctca acgacaaggttattcagttt tgtgaaaact cgggaattaa 2640 gattaaggaa tatttgatgc actatacaagaaaagaagat tgggttaaac attttggacc 2700 aaaatgggat gattttttaa gaaagaaaattatgtttgat cccaaaagac tattgtctcc 2760 aggacaagac atatttaatt aa 2782 8524 PRT Arabidopsis thaliana 8 Met Thr Asn Thr Leu Cys Leu Ser Leu IleThr Leu Ile Thr Leu Phe 1 5 10 15 Ile Ser Leu Thr Pro Thr Leu Ile LysSer Asp Glu Gly Ile Asp Val 20 25 30 Phe Leu Pro Ile Ser Leu Asn Leu ThrVal Leu Thr Asp Pro Phe Ser 35 40 45 Ile Ser Ala Ala Ser His Asp Phe GlyAsn Ile Thr Asp Glu Asn Pro 50 55 60 Gly Ala Val Leu Cys Pro Ser Ser ThrThr Glu Val Ala Arg Leu Leu 65 70 75 80 Arg Phe Ala Asn Gly Gly Phe SerTyr Asn Lys Gly Ser Thr Ser Pro 85 90 95 Ala Ser Thr Phe Lys Val Ala AlaArg Gly Gln Gly His Ser Leu Arg 100 105 110 Gly Gln Ala Ser Ala Pro GlyGly Val Val Val Asn Met Thr Cys Leu 115 120 125 Ala Met Ala Ala Lys ProAla Ala Val Val Ile Ser Ala Asp Gly Thr 130 135 140 Tyr Ala Asp Val AlaAla Gly Thr Met Trp Val Asp Val Leu Lys Ala 145 150 155 160 Ala Val AspArg Gly Val Ser Pro Val Thr Trp Thr Asp Tyr Leu Tyr 165 170 175 Leu SerVal Gly Gly Thr Leu Ser Asn Ala Gly Ile Gly Gly Gln Thr 180 185 190 PheArg His Gly Pro Gln Ile Ser Asn Val His Glu Leu Asp Val Ile 195 200 205Thr Gly Lys Gly Glu Met Met Thr Cys Ser Pro Lys Leu Asn Pro Glu 210 215220 Leu Phe Tyr Gly Val Leu Gly Gly Leu Gly Gln Phe Gly Ile Ile Thr 225230 235 240 Arg Ala Arg Ile Ala Leu Asp His Ala Pro Thr Arg Val Lys TrpSer 245 250 255 Arg Ile Leu Tyr Ser Asp Phe Ser Ala Phe Lys Arg Asp GlnGlu Arg 260 265 270 Leu Ile Ser Met Thr Asn Asp Leu Gly Val Asp Phe LeuGlu Gly Gln 275 280 285 Leu Met Met Ser Asn Gly Phe Val Asp Thr Ser PhePhe Pro Leu Ser 290 295 300 Asp Gln Thr Arg Val Ala Ser Leu Val Asn AspHis Arg Ile Ile Tyr 305 310 315 320 Val Leu Glu Val Ala Lys Tyr Tyr AspArg Thr Thr Leu Pro Ile Ile 325 330 335 Asp Gln Val Ile Asp Thr Leu SerArg Thr Leu Gly Phe Ala Pro Gly 340 345 350 Phe Met Phe Val Gln Asp ValPro Tyr Phe Asp Phe Leu Asn Arg Val 355 360 365 Arg Asn Glu Glu Asp LysLeu Arg Ser Leu Gly Leu Trp Glu Val Pro 370 375 380 His Pro Trp Leu AsnIle Phe Val Pro Gly Ser Arg Ile Gln Asp Phe 385 390 395 400 His Asp GlyVal Ile Asn Gly Leu Leu Leu Asn Gln Thr Ser Thr Ser 405 410 415 Gly ValThr Leu Phe Tyr Pro Thr Asn Arg Asn Lys Trp Asn Asn Arg 420 425 430 MetSer Thr Met Thr Pro Asp Glu Asp Val Phe Tyr Val Ile Gly Leu 435 440 445Leu Gln Ser Ala Gly Gly Ser Gln Asn Trp Gln Glu Leu Glu Asn Leu 450 455460 Asn Asp Lys Val Ile Gln Phe Cys Glu Asn Ser Gly Ile Lys Ile Lys 465470 475 480 Glu Tyr Leu Met His Tyr Thr Arg Lys Glu Asp Trp Val Lys HisPhe 485 490 495 Gly Pro Lys Trp Asp Asp Phe Leu Arg Lys Lys Ile Met PheAsp Pro 500 505 510 Lys Arg Leu Leu Ser Pro Gly Gln Asp Ile Phe Asn 515520 9 2805 DNA Arabidopsis thaliana 9 atgacgtcaa gctttcttct cctgacgttcgccatatgta aactgatcat agccgtgggt 60 ctaaacgtgg gccccagtga gctcctccgcatcggagcca tagatgtcga cggccacttc 120 accgtccacc cttccgactt agcctccgtctcctcagact tcggtatgct gaagtcacct 180 gaagagccat tggccgtgct tcatccatcatcggccgaag acgtggcacg actcgtcaga 240 acagcttacg gttcagccac ggcgtttccggtctcagccc gaggccacgg ccattccata 300 aacggacaag ccgcggcggg gaggaacggtgtggtggttg aaatgaacca cggcgtaacc 360 gggacgccca agccactcgt ccgaccggatgaaatgtatg tggatgtatg gggtggagag 420 ttatgggtcg atgtgttgaa gaaaacgttggagcatggct tagcaccaaa atcatggacg 480 gattacttgt atctaaccgt tggaggtacactctccaatg caggaatcag tggtcaagct 540 tttcaccatg gtcctcaaat tagtaacgtccttgagctcg acgttgtaac tggttagtat 600 taaaacattc aagttcatat attttaaatgcttttgtctg aagttttact aataacaaga 660 aattgatacc aaaaagtagg gaaaggagaggtgatgagat gctcagaaga agagaacaca 720 aggctattcc atggagttct tggtggattaggtcaatttg ggatcatcac tcgagcacga 780 atctctctcg aaccagctcc ccaaagggtaatattttttt aatgactagc tatcaaaaat 840 ccctggcggg tccatacgtt gtaatctttttagtttttac tgttgatggt attttttata 900 tattttggat aataaaaccc taaaatggtatattgtgatg acaggtgaga tggatacggg 960 tattgtattc gagcttcaaa gtgtttacggaggaccaaga gtacttaatc tcaatgcatg 1020 gtcaattaaa gtttgattac gtggaaggttttgtgattgt ggacgaagga ctcgtcaaca 1080 attggagatc ttctttcttc tctccacgtaaccccgtcaa gatctcctct gttagttcca 1140 acggctctgt tttgtattgc cttgagatcaccaagaacta ccacgactcc gactccgaaa 1200 tcgttgatca ggtcactttc attattcacttagaaaaaag cgatattttc attttttata 1260 ttgatgaata tctggaagga tttaacgctatgcgactatt gggaaatcat tatgaaaaaa 1320 tatttagttt atatgattga aagtggtctccatagtattt ttgttgtgtc gactttatta 1380 taacttaaat ttggaagagg acatgaagaagaagccagag aggatctaca gagatctagc 1440 ttttccacct gaacttaata atgcacatttatataattat ttttcttctt ctaaagttta 1500 gtttatcact agcgaattaa tcatggttactaattaagta gtggacaggg tcatggacca 1560 ctcactcacc aaataatgat tcctctttactcttaagttt aattttaata aaaccaactc 1620 tactggaatc ttaacttatc cttggttttggtaggctttt atagcaacac ggttttttta 1680 attttcctat tccagatttt gtatattaaatgtcgatttt ttttcttttt gtttcaggaa 1740 gttgagattc tgatgaagaa attgaatttcataccgacat cggtctttac aacggattta 1800 caatatgtgg actttctcga ccgggtacacaaggccgaat tgaagctccg gtccaagaat 1860 ttatgggagg ttccacaccc atggctcaacctcttcgtgc caaaatcaag aatctctgac 1920 ttcgataaag gcgttttcaa gggcattttgggaaataaaa caagtggccc tattcttatc 1980 taccccatga acaaagacaa gtaagtcttgacattaccat tgattactac ttctaaattt 2040 cttctctaga aaaaagaata aaacgagttttgcattgcat gcatgcaaag ttacacttgt 2100 ggggattaat tagtggtcca agaaaaaaagtttgtcaaaa ttgaaaaaaa ctagacacgt 2160 ggtacatggg attgtccgaa aaacgttgtccacatgtgca tcgaaccagc taagattgac 2220 aacaacactt cgtcggctcg tatttctctttttgttttgt gaccaaatcc gatggtccag 2280 attgggttta tttgttttta agttcctagaactcatggtg ggtgggtccc aatcagattc 2340 tcctagacca aaccgatctc aacgaaccctccgcacatca ttgattatta cattaatata 2400 gatattgtcg ttgctgacgt gtcgtaatttgatgttattg tcagatggga cgagaggagc 2460 tcagccgtga cgccggatga ggaagttttctatctggtgg ctctattgag atcagcttta 2520 acggacggtg aagagacaca gaagctagagtatctgaaag atcagaaccg tcggatcttg 2580 gagttctgtg aacaagccaa gatcaatgtgaagcagtatc ttcctcacca cgcaacacag 2640 gaagagtggg tggctcattt tggggacaagtgggatcggt tcagaagctt aaaggctgag 2700 tttgatccgc gacacatact cgctactggtcagagaatct ttcaaaaccc atctttgtct 2760 ttgtttcctc cgtcgtcgtc ttcttcgtcagcggcttcat ggtga 2805 10 536 PRT Arabidopsis thaliana 10 Met Thr Ser SerPhe Leu Leu Leu Thr Phe Ala Ile Cys Lys Leu Ile 1 5 10 15 Ile Ala ValGly Leu Asn Val Gly Pro Ser Glu Leu Leu Arg Ile Gly 20 25 30 Ala Ile AspVal Asp Gly His Phe Thr Val His Pro Ser Asp Leu Ala 35 40 45 Ser Val SerSer Asp Phe Gly Met Leu Lys Ser Pro Glu Glu Pro Leu 50 55 60 Ala Val LeuHis Pro Ser Ser Ala Glu Asp Val Ala Arg Leu Val Arg 65 70 75 80 Thr AlaTyr Gly Ser Ala Thr Ala Phe Pro Val Ser Ala Arg Gly His 85 90 95 Gly HisSer Ile Asn Gly Gln Ala Ala Ala Gly Arg Asn Gly Val Val 100 105 110 ValGlu Met Asn His Gly Val Thr Gly Thr Pro Lys Pro Leu Val Arg 115 120 125Pro Asp Glu Met Tyr Val Asp Val Trp Gly Gly Glu Leu Trp Val Asp 130 135140 Val Leu Lys Lys Thr Leu Glu His Gly Leu Ala Pro Lys Ser Trp Thr 145150 155 160 Asp Tyr Leu Tyr Leu Thr Val Gly Gly Thr Leu Ser Asn Ala GlyIle 165 170 175 Ser Gly Gln Ala Phe His His Gly Pro Gln Ile Ser Asn ValLeu Glu 180 185 190 Leu Asp Val Val Thr Gly Lys Gly Glu Val Met Arg CysSer Glu Glu 195 200 205 Glu Asn Thr Arg Leu Phe His Gly Val Leu Gly GlyLeu Gly Gln Phe 210 215 220 Gly Ile Ile Thr Arg Ala Arg Ile Ser Leu GluPro Ala Pro Gln Arg 225 230 235 240 Val Arg Trp Ile Arg Val Leu Tyr SerSer Phe Lys Val Phe Thr Glu 245 250 255 Asp Gln Glu Tyr Leu Ile Ser MetHis Gly Gln Leu Lys Phe Asp Tyr 260 265 270 Val Glu Gly Phe Val Ile ValAsp Glu Gly Leu Val Asn Asn Trp Arg 275 280 285 Ser Ser Phe Phe Ser ProArg Asn Pro Val Lys Ile Ser Ser Val Ser 290 295 300 Ser Asn Gly Ser ValLeu Tyr Cys Leu Glu Ile Thr Lys Asn Tyr His 305 310 315 320 Asp Ser AspSer Glu Ile Val Asp Gln Glu Val Glu Ile Leu Met Lys 325 330 335 Lys LeuAsn Phe Ile Pro Thr Ser Val Phe Thr Thr Asp Leu Gln Tyr 340 345 350 ValAsp Phe Leu Asp Arg Val His Lys Ala Glu Leu Lys Leu Arg Ser 355 360 365Lys Asn Leu Trp Glu Val Pro His Pro Trp Leu Asn Leu Phe Val Pro 370 375380 Lys Ser Arg Ile Ser Asp Phe Asp Lys Gly Val Phe Lys Gly Ile Leu 385390 395 400 Gly Asn Lys Thr Ser Gly Pro Ile Leu Ile Tyr Pro Met Asn LysAsp 405 410 415 Lys Trp Asp Glu Arg Ser Ser Ala Val Thr Pro Asp Glu GluVal Phe 420 425 430 Tyr Leu Val Ala Leu Leu Arg Ser Ala Leu Thr Asp GlyGlu Glu Thr 435 440 445 Gln Lys Leu Glu Tyr Leu Lys Asp Gln Asn Arg ArgIle Leu Glu Phe 450 455 460 Cys Glu Gln Ala Lys Ile Asn Val Lys Gln TyrLeu Pro His His Ala 465 470 475 480 Thr Gln Glu Glu Trp Val Ala His PheGly Asp Lys Trp Asp Arg Phe 485 490 495 Arg Ser Leu Lys Ala Glu Phe AspPro Arg His Ile Leu Ala Thr Gly 500 505 510 Gln Arg Ile Phe Gln Asn ProSer Leu Ser Leu Phe Pro Pro Ser Ser 515 520 525 Ser Ser Ser Ser Ala AlaSer Trp 530 535 11 1936 DNA Arabidopsis thaliana 11 atgcttatagtaagaagttt caccatcttg cttctcagct gcatagcctt taagttggct 60 tgctgcttctctagcagcat ttcttctttg aaggcgcttc ccctagtagg ccatttggag 120 tttgaacatgtccatcacgc ctccaaagat tttggaaatc gataccagtt gatccctttg 180 gcggtcttacatcccaaatc ggtaagcgac atcgcctcaa cgatacgaca catctggatg 240 atgggcactcattcacagct tacagtggca gcgagaggtc gtggacattc actccaaggc 300 caagctcaaacaagacatgg aattgttata cacatggaat cactccatcc ccagaagctg 360 caggtctacagtgtggattc ccctgctcca tatgttgatg tgtctggtgg tgagctgtgg 420 ataaacattttgcatgagac cctcaagtac gggcttgcac caaaatcatg gacggattac 480 ctgcatttaactgtaggtgg tactctgtcc aatgctggaa taagcggcca ggcattccga 540 catggaccacagatcagcaa tgttcatcaa ctggagattg tcacaggtta gttcagagtt 600 gcagtattcgtgttttgaaa gcatagactc tatatggttg gtgactatta acaacatgaa 660 gagattcccgagaatagcta cccactaatg tcatgcctat ttattgactg caggaaaagg 720 cgagatcctaaactgtacaa agaggcagaa cagcgactta tttaatggtg ttcttggtgg 780 tttaggtcagtttggcatca taacgcgggc aagaatagca ttggaaccag caccaaccat 840 ggtaaacaataaataaataa aaaacttaaa aactgaacac gcgtgtgtcc tcctaactct 900 gtataatggacaggtaaaat ggataagagt gttatacctg gattttgcag cttttgccaa 960 ggaccaagagcaactaatat ctgcccaggg ccacaaattc gattacatag aagggtttgt 1020 gataataaacaggacaggcc tcctgaacag ctggaggttg tctttcaccg cagaagagcc 1080 tttagaagcaagccaattca agtttgatgg aaggactctg tattgtctgg agctagccaa 1140 gtatttgaagcaagataaca aagacgtaat caaccaggtg agaaaacaga gtagaagcaa 1200 tcggtagaatcttctttggt agatgacatt cattggaact gaaaatatat atatatttgt 1260 ccaatccaggaagtgaaaga aacattatca gagctaagct acgtgacgtc gacactgttt 1320 acaacggaggtagcatatga agcattcttg gacagggtac atgtgtctga ggtaaaactc 1380 cgatcgaaagggcagtggga ggtgccacat ccatggctga acctcctggt accaagaagc 1440 aaaatcaatgaatttgcaag aggtgtattt ggaaacatac taacggatac aagcaacggc 1500 ccagtcatcgtctacccagt gaacaaatca aagtaagaaa gaaagaaaga aagagctagt 1560 catgattttgtttcttttca cttgttgaca aaacaaaagc atgttggtga gcaggtggga 1620 caatcaaacatcagcagtaa caccggagga agaggtattc tacctggtgg cgatcctaac 1680 atcggcatctccagggtcgg caggaaagga tggagtagaa gagatcttga ggcggaacag 1740 aagaatactggaattcagtg aagaagcagg gatagggttg aagcagtatc tgccacatta 1800 cacgacaagagaagagtgga gatcccattt cggggacaag tggggagaat ttgtgaggag 1860 gaaatccagatatgatccat tggcaattct tgcgcctggc caccgaattt ttcaaaaggc 1920 agtctcatactcatga 1936 12 504 PRT Arabidopsis thaliana 12 Met Leu Ile Val Arg SerPhe Thr Ile Leu Leu Leu Ser Cys Ile Ala 1 5 10 15 Phe Lys Leu Ala CysCys Phe Ser Ser Ser Ile Ser Ser Leu Lys Ala 20 25 30 Leu Pro Leu Val GlyHis Leu Glu Phe Glu His Val His His Ala Ser 35 40 45 Lys Asp Phe Gly AsnArg Tyr Gln Leu Ile Pro Leu Ala Val Leu His 50 55 60 Pro Lys Ser Val SerAsp Ile Ala Ser Thr Ile Arg His Ile Trp Met 65 70 75 80 Met Gly Thr HisSer Gln Leu Thr Val Ala Ala Arg Gly Arg Gly His 85 90 95 Ser Leu Gln GlyGln Ala Gln Thr Arg His Gly Ile Val Ile His Met 100 105 110 Glu Ser LeuHis Pro Gln Lys Leu Gln Val Tyr Ser Val Asp Ser Pro 115 120 125 Ala ProTyr Val Asp Val Ser Gly Gly Glu Leu Trp Ile Asn Ile Leu 130 135 140 HisGlu Thr Leu Lys Tyr Gly Leu Ala Pro Lys Ser Trp Thr Asp Tyr 145 150 155160 Leu His Leu Thr Val Gly Gly Thr Leu Ser Asn Ala Gly Ile Ser Gly 165170 175 Gln Ala Phe Arg His Gly Pro Gln Ile Ser Asn Val His Gln Leu Glu180 185 190 Ile Val Thr Gly Lys Gly Glu Ile Leu Asn Cys Thr Lys Arg GlnAsn 195 200 205 Ser Asp Leu Phe Asn Gly Val Leu Gly Gly Leu Gly Gln PheGly Ile 210 215 220 Ile Thr Arg Ala Arg Ile Ala Leu Glu Pro Ala Pro ThrMet Asp Gln 225 230 235 240 Glu Gln Leu Ile Ser Ala Gln Gly His Lys PheAsp Tyr Ile Glu Gly 245 250 255 Phe Val Ile Ile Asn Arg Thr Gly Leu LeuAsn Ser Trp Arg Leu Ser 260 265 270 Phe Thr Ala Glu Glu Pro Leu Glu AlaSer Gln Phe Lys Phe Asp Gly 275 280 285 Arg Thr Leu Tyr Cys Leu Glu LeuAla Lys Tyr Leu Lys Gln Asp Asn 290 295 300 Lys Asp Val Ile Asn Gln GluVal Lys Glu Thr Leu Ser Glu Leu Ser 305 310 315 320 Tyr Val Thr Ser ThrLeu Phe Thr Thr Glu Val Ala Tyr Glu Ala Phe 325 330 335 Leu Asp Arg ValHis Val Ser Glu Val Lys Leu Arg Ser Lys Gly Gln 340 345 350 Trp Glu ValPro His Pro Trp Leu Asn Leu Leu Val Pro Arg Ser Lys 355 360 365 Ile AsnGlu Phe Ala Arg Gly Val Phe Gly Asn Ile Leu Thr Asp Thr 370 375 380 SerAsn Gly Pro Val Ile Val Tyr Pro Val Asn Lys Ser Lys Trp Asp 385 390 395400 Asn Gln Thr Ser Ala Val Thr Pro Glu Glu Glu Val Phe Tyr Leu Val 405410 415 Ala Ile Leu Thr Ser Ala Ser Pro Gly Ser Ala Gly Lys Asp Gly Val420 425 430 Glu Glu Ile Leu Arg Arg Asn Arg Arg Ile Leu Glu Phe Ser GluGlu 435 440 445 Ala Gly Ile Gly Leu Lys Gln Tyr Leu Pro His Tyr Thr ThrArg Glu 450 455 460 Glu Trp Arg Ser His Phe Gly Asp Lys Trp Gly Glu PheVal Arg Arg 465 470 475 480 Lys Ser Arg Tyr Asp Pro Leu Ala Ile Leu AlaPro Gly His Arg Ile 485 490 495 Phe Gln Lys Ala Val Ser Tyr Ser 500 1331 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide primer or probe 13 cggtcgacat gggattgacc tcatccttac g 3114 35 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide primer or probe 14 gcgtcgactt atacagttct aggtttcggcagtat 35 15 33 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide primer or probe 15 gcggtaccag agagagaaacataaacaaat ggc 33 16 31 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide primer or probe 16 gcggtacccaattttacttc caccaaaatg c 31 17 34 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide primer or probe 17 gcggtaccttcattgataag aatcaagcta ttca 34 18 31 DNA Artificial Sequence Descriptionof Artificial Sequence oligonucleotide primer or probe 18 gcggtacccaaagtggtgag aacgactaac a 31 19 28 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide primer or probe 19 gcggtacccccattaaccta cccgtttg 28 20 32 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide primer or probe 20 gcggtaccagacgatgaacg tacttgtctg ta 32 21 28 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide primer or probe 21 ggggtaccttgatgaatcgt gaaatgac 28 22 31 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide primer or probe 22 ggggtaccctttcctcttgg ttttgtcctg t 31 23 32 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide primer or probe 23 gctctagatcaggaaaagaa ccatgcttat ag 32 24 32 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide primer or probe 24 gctctagatcatgagtatga gactgccttt tg 32 25 1728 DNA Arabidopsis thaliana 25atgggattga cctcatcctt acggttccat agacaaaaca acaagacttt cctcggaatc 60ttcatgatct tagttctaag ctgtatacca ggtagaacca atctttgttc caatcattct 120gttagtaccc caaaagaatt accttcttca aatccttcag atattcgttc ctcattagtt 180tcactagatt tggagggtta tataagcttc gacgatgtcc acaatgtggc caaggacttt 240ggcaacagat accagttacc acctttggca attctacatc caaggtcagt ttttgatatt 300tcatcgatga tgaagcatat agtacatctg ggctccacct caaatcttac agtagcagct 360agaggccatg gtcactcgct tcaaggacaa gctctagctc atcaaggtgt tgtcatcaaa 420atggagtcac ttcgaagtcc tgatatcagg atttataagg ggaagcaacc atatgttgat 480gtctcaggtg gtgaaatatg gataaacatt ctacgcgaga ctctaaaata cggtctttca 540ccaaagtcct ggacagacta ccttcatttg accgttggag gtacactatc taatgctgga 600atcagcggtc aagcattcaa gcatggaccc caaatcaaca acgtctacca gctagagatt 660gttacaggga aaggagaagt cgtaacctgt tctgagaagc ggaattctga acttttcttc 720agtgttcttg gcgggcttgg acagtttggc ataatcaccc gggcacggat ctctcttgaa 780ccagcaccgc atatggttaa atggatcagg gtactctact ctgacttttc tgcattttca 840agggaccaag aatatctgat ttcgaaggag aaaacttttg attacgttga aggatttgtg 900ataatcaata gaacagacct tctcaataat tggcgatcgt cattcagtcc caacgattcc 960acacaggcaa gcagattcaa gtcagatggg aaaactcttt attgcctaga agtggtcaaa 1020tatttcaacc cagaagaagc tagctctatg gatcaggaaa ctggcaagtt actttcagag 1080ttaaattata ttccatccac tttgttttca tctgaagtgc catatatcga gtttctggat 1140cgcgtgcata tcgcagagag aaaactaaga gcaaagggtt tatgggaggt tccacatccc 1200tggctgaatc tcctgattcc taagagcagc atataccaat ttgctacaga agttttcaac 1260aacattctca caagcaacaa caacggtcct atccttattt atccagtcaa tcaatccaag 1320tggaagaaac atacatcttt gataactcca aatgaagata tattctatct cgtagccttt 1380ctcccctctg cagtgccaaa ttcctcaggg aaaaacgatc tagagtacct tttgaaacaa 1440aaccaaagag ttatgaactt ctgcgcagca gcaaacctca acgtgaagca gtatttgccc 1500cattatgaaa ctcaaaaaga gtggaaatca cactttggca aaagatggga aacatttgca 1560cagaggaaac aagcctacga ccctctagcg attctagcac ctggccaaag aatattccaa 1620aagacaacag gaaaattatc tcccatccaa ctcgcaaagt caaaggcaac aggaagtcct 1680caaaggtacc attacgcatc aatactgccg aaacctagaa ctgtataa 1728 26 1506 DNAArabidopsis thaliana 26 atggctaatc ttcgtttaat gatcacttta atcacggttttaatgatcac caaatcatca 60 aacggtatta aaattgattt acctaaatcc cttaacctcaccctctctac cgatccttcc 120 atcatctccg cagcctctca tgacttcgga aacataaccaccgtgacccc cggcggcgta 180 atctgcccct cctccaccgc tgatatctct cgtctcctccaatacgccgc aaacggaaaa 240 agtacattcc aagtagcggc tcgtggccaa ggccactccttaaacggcca agcctcggtc 300 tccggcggag taatcgtcaa catgacgtgt atcactgacgtggtggtttc aaaagacaag 360 aagtacgctg acgtggcggc cgggacgtta tgggtggatgtgcttaagaa gacggcggag 420 aaaggggtgt cgccggtttc ttggacggat tatttgcatataaccgtcgg aggaacgttg 480 tcgaatggtg gaattggtgg tcaagtgttt cgaaacggtcctcttgttag taacgtcctt 540 gaattggacg ttattactgg gaaaggtgaa atgttgacatgctcgcgaca gctaaaccca 600 gaattgttct atggagtgtt aggaggtttg ggtcaatttggaattataac gagagccaga 660 attgttttgg accatgcacc taaacgggcc aaatggtttcggatgctcta cagtgatttc 720 acaactttta caaaggacca agaacgtttg atatcaatggcaaacgatat tggagtcgac 780 tatttagaag gtcaaatatt tctatcaaac ggtgtcgttgacacctcttt tttcccacct 840 tcagatcaat ctaaagtcgc tgatctagtc aagcaacacggtatcatcta tgttcttgaa 900 gtagccaagt attatgatga tcccaatctc cccatcatcagcaaggttat tgacacatta 960 acgaaaacat taagttactt gcccgggttc atatcaatgcacgacgtggc ctacttcgat 1020 ttcttgaacc gtgtacatgt cgaagaaaat aaactcagatctttgggatt atgggaactt 1080 cctcatcctt ggcttaacct ctacgttcct aaatctcggattctcgattt tcataacggt 1140 gttgtcaaag acattcttct taagcaaaaa tcagcttcgggactcgctct tctctatcca 1200 acaaaccgga ataaatggga caatcgtatg tcggcgatgataccagagat cgatgaagat 1260 gttatatata ttatcggact actacaatcc gctaccccaaaggatcttcc agaagtggag 1320 agcgttaacg agaagataat taggttttgc aaggattcaggtattaagat taagcaatat 1380 ctaatgcatt atactagtaa agaagattgg attgagcattttggatcaaa atgggatgat 1440 ttttcgaaga ggaaagatct atttgatccc aagaaactgttatctccagg gcaagacatc 1500 ttttga 1506 27 1572 DNA Arabidopsis thaliana27 atggcgagtt ataatcttcg ttcacaagtt cgtcttatag caataacaat agtaatcatc 60attactctct caactccgat cacaaccaac acatcaccac aaccatggaa tatcctttca 120cacaacgaat tcgccggaaa actcacctcc tcctcctcct ccgtcgaatc agccgccaca 180gatttcggcc acgtcaccaa aatcttccct tccgccgtct taatcccttc ctccgttgaa 240gacatcacag atctcataaa actctctttt gactctcaac tgtcttttcc tttagccgct 300cgtggtcacg gacacagcca ccgtggccaa gcctcggcta aagacggagt tgtggtcaac 360atgcggtcca tggtaaaccg ggatcgaggt atcaaggtgt ctaggacctg tttatatgtt 420gacgtggacg ctgcgtggct atggattgag gtgttgaata aaactttgga gttagggtta 480acgccggttt cttggacgga ttatttgtat ttaacagtcg gtgggacgtt atcaaacggc 540ggaattagtg gacaaacgtt tcggtacggt ccacagatca ctaatgttct agagatggat 600gttattactg gaaaaggaga gattgcaact tgttccaagg acatgaactc ggatcttttc 660ttcgcggtgt taggaggttt gggtcaattc ggcattataa caagagccag aattaaactt 720gaagtagctc cgaaaagggc caagtggtta aggtttctat acatagattt ctccgaattc 780acaagagatc aagaacgagt gatatcgaaa acggacggtg tagatttctt agaaggttcc 840attatggtgg accatggccc accggataac tggagatcca cgtattatcc accgtccgat 900cacttgagga tcgcctcaat ggtcaaacga catcgtgtca tctactgcct tgaagtcgtc 960aagtattacg acgaaacttc tcaatacaca gtcaacgagg aaatggagga gttaagcgat 1020agtttaaacc atgtaagagg gtttatgtac gagaaagatg tgacgtatat ggatttccta 1080aaccgagttc gaaccggaga gctaaacctg aaatccaaag gccaatggga tgttccacat 1140ccatggctta atctcttcgt accaaaaact caaatctcca aatttgatga tggtgttttt 1200aagggtatta tcctaagaaa taacatcact agcggtcctg ttcttgttta tcctatgaat 1260cgcaacaagt ggaatgatcg gatgtctgcc gctatacccg aggaagatgt attttatgcg 1320gtagggtttt taagatccgc gggttttgac aattgggagg cttttgatca agaaaacatg 1380gaaatactga agttttgtga ggatgctaat atgggggtta tacaatatct tccttatcat 1440tcatcacaag aaggatgggt tagacatttt ggtccgaggt ggaatatttt cgtagagaga 1500aaatataaat atgatcccaa aatgatatta tcaccgggac aaaatatatt tcaaaaaata 1560aactcgagtt ag 1572 28 1575 DNA Arabidopsis thaliana 28 atgactaatactctctgttt aagcctcatc accctaataa cgctttttat aagtttaacc 60 ccaaccttaatcaaatcaga tgagggcatt gatgttttct tacccatatc actcaacctt 120 acggtcctaaccgatccctt ctccatctct gccgcttctc acgacttcgg taacataacc 180 gacgaaaatcccggcgccgt cctctgccct tcctccacca cggaggtggc tcgtctcctc 240 cgtttcgctaacggaggatt ctcttacaat aaaggctcaa ccagccccgc gtctactttc 300 aaagtggctgctcgaggcca aggccactcc ctccgtggcc aagcctctgc acccggaggt 360 gtcgtcgtgaacatgacgtg tctcgccatg gcggctaaac cagcggcggt tgttatctcg 420 gcagacgggacttacgctga cgtggctgcc gggacgatgt gggtggatgt tctgaaggcg 480 gcggtggatagaggcgtctc gccggttaca tggacggatt atttgtatct cagcgtcggc 540 gggacgttgtcgaacgctgg aatcggtggt cagacgttta gacacggccc tcagattagt 600 aacgttcatgagcttgacgt tattaccgga aaaggtgaaa tgatgacttg ctctccaaag 660 ttaaaccctgaattgttcta tggagtttta ggaggtttgg gtcaattcgg tattataacg 720 agggccaggattgcgttgga tcatgcaccc acaagggtga aatggtctcg catactctac 780 agtgacttctcggcttttaa aagagaccaa gagcgtttaa tatcaatgac caatgatctc 840 ggagttgactttttggaagg tcaacttatg atgtcaaatg gcttcgtaga cacctctttc 900 ttcccactctccgatcaaac aagagtcgca tctcttgtga atgaccaccg gatcatctat 960 gttctcgaagtagccaagta ttatgacaga accacccttc ccattattga ccaggtgatt 1020 gacacgttaagtagaactct aggtttcgct ccagggttta tgttcgtaca agatgttccg 1080 tatttcgatttcttgaaccg tgtccgaaac gaagaagata aactcagatc tttaggacta 1140 tgggaagttcctcatccatg gcttaacatc tttgtcccgg ggtctcgaat ccaagatttt 1200 catgatggtgttattaatgg ccttcttcta aaccaaacct caacttctgg tgttactctc 1260 ttctatcccacaaaccgaaa caaatggaac aaccgcatgt caacgatgac accggacgaa 1320 gatgttttttatgtgatcgg attactgcaa tcagctggtg gatctcaaaa ttggcaagaa 1380 cttgaaaatctcaacgacaa ggttattcag ttttgtgaaa actcgggaat taagattaag 1440 gaatatttgatgcactatac aagaaaagaa gattgggtta aacattttgg accaaaatgg 1500 gatgattttttaagaaagaa aattatgttt gatcccaaaa gactattgtc tccaggacaa 1560 gacatatttaattaa 1575 29 1611 DNA Arabidopsis thaliana 29 atgacgtcaa gctttcttctcctgacgttc gccatatgta aactgatcat agccgtgggt 60 ctaaacgtgg gccccagtgagctcctccgc atcggagcca tagatgtcga cggccacttc 120 accgtccacc cttccgacttagcctccgtc tcctcagact tcggtatgct gaagtcacct 180 gaagagccat tggccgtgcttcatccatca tcggccgaag acgtggcacg actcgtcaga 240 acagcttacg gttcagccacggcgtttccg gtctcagccc gaggccacgg ccattccata 300 aacggacaag ccgcggcggggaggaacggt gtggtggttg aaatgaacca cggcgtaacc 360 gggacgccca agccactcgtccgaccggat gaaatgtatg tggatgtatg gggtggagag 420 ttatgggtcg atgtgttgaagaaaacgttg gagcatggct tagcaccaaa atcatggacg 480 gattacttgt atctaaccgttggaggtaca ctctccaatg caggaatcag tggtcaagct 540 tttcaccatg gtcctcaaattagtaacgtc cttgagctcg acgttgtaac tgggaaagga 600 gaggtgatga gatgctcagaagaagagaac acaaggctat tccatggagt tcttggtgga 660 ttaggtcaat ttgggatcatcactcgagca cgaatctctc tcgaaccagc tccccaaagg 720 gtgagatgga tacgggtattgtattcgagc ttcaaagtgt ttacggagga ccaagagtac 780 ttaatctcaa tgcatggtcaattaaagttt gattacgtgg aaggttttgt gattgtggac 840 gaaggactcg tcaacaattggagatcttct ttcttctctc cacgtaaccc cgtcaagatc 900 tcctctgtta gttccaacggctctgttttg tattgccttg agatcaccaa gaactaccac 960 gactccgact ccgaaatcgttgatcaggaa gttgagattc tgatgaagaa attgaatttc 1020 ataccgacat cggtctttacaacggattta caatatgtgg actttctcga ccgggtacac 1080 aaggccgaat tgaagctccggtccaagaat ttatgggagg ttccacaccc atggctcaac 1140 ctcttcgtgc caaaatcaagaatctctgac ttcgataaag gcgttttcaa gggcattttg 1200 ggaaataaaa caagtggccctattcttatc taccccatga acaaagacaa atgggacgag 1260 aggagctcag ccgtgacgccggatgaggaa gttttctatc tggtggctct attgagatca 1320 gctttaacgg acggtgaagagacacagaag ctagagtatc tgaaagatca gaaccgtcgg 1380 atcttggagt tctgtgaacaagccaagatc aatgtgaagc agtatcttcc tcaccacgca 1440 acacaggaag agtgggtggctcattttggg gacaagtggg atcggttcag aagcttaaag 1500 gctgagtttg atccgcgacacatactcgct actggtcaga gaatctttca aaacccatct 1560 ttgtctttgt ttcctccgtcgtcgtcttct tcgtcagcgg cttcatggtg a 1611 30 1515 DNA Arabidopsis thaliana30 atgcttatag taagaagttt caccatcttg cttctcagct gcatagcctt taagttggct 60tgctgcttct ctagcagcat ttcttctttg aaggcgcttc ccctagtagg ccatttggag 120tttgaacatg tccatcacgc ctccaaagat tttggaaatc gataccagtt gatccctttg 180gcggtcttac atcccaaatc ggtaagcgac atcgcctcaa cgatacgaca catctggatg 240atgggcactc attcacagct tacagtggca gcgagaggtc gtggacattc actccaaggc 300caagctcaaa caagacatgg aattgttata cacatggaat cactccatcc ccagaagctg 360caggtctaca gtgtggattc ccctgctcca tatgttgatg tgtctggtgg tgagctgtgg 420ataaacattt tgcatgagac cctcaagtac gggcttgcac caaaatcatg gacggattac 480ctgcatttaa ctgtaggtgg tactctgtcc aatgctggaa taagcggcca ggcattccga 540catggaccac agatcagcaa tgttcatcaa ctggagattg tcacaggaaa aggcgagatc 600ctaaactgta caaagaggca gaacagcgac ttatttaatg gtgttcttgg tggtttaggt 660cagtttggca tcataacgcg ggcaagaata gcattggaac cagcaccaac catggaccaa 720gagcaactaa tatctgccca gggccacaaa ttcgattaca tagaagggtt tgtgataata 780aacaggacag gcctcctgaa cagctggagg ttgtctttca ccgcagaaga gcctttagaa 840gcaagccaat tcaagtttga tggaaggact ctgtattgtc tggagctagc caagtatttg 900aagcaagata acaaagacgt aatcaaccag gaagtgaaag aaacattatc agagctaagc 960tacgtgacgt cgacactgtt tacaacggag gtagcatatg aagcattctt ggacagggta 1020catgtgtctg aggtaaaact ccgatcgaaa gggcagtggg aggtgccaca tccatggctg 1080aacctcctgg taccaagaag caaaatcaat gaatttgcaa gaggtgtatt tggaaacata 1140ctaacggata caagcaacgg cccagtcatc gtctacccag tgaacaaatc aaagtgggac 1200aatcaaacat cagcagtaac accggaggaa gaggtattct acctggtggc gatcctaaca 1260tcggcatctc cagggtcggc aggaaaggat ggagtagaag agatcttgag gcggaacaga 1320agaatactgg aattcagtga agaagcaggg atagggttga agcagtatct gccacattac 1380acgacaagag aagagtggag atcccatttc ggggacaagt ggggagaatt tgtgaggagg 1440aaatccagat atgatccatt ggcaattctt gcgcctggcc accgaatttt tcaaaaggca 1500gtctcatact catga 1515 31 84 DNA Arabidopsis thaliana 31 tcagcttcgggactcgctct tctctatcca acaaaccgga ataaatggga caatcgtatg 60 tcggcgatgataccagagat cgat 84 32 28 PRT Arabidopsis thaliana 32 Ser Ala Ser Gly LeuAla Leu Leu Tyr Pro Thr Asn Arg Asn Lys Trp 1 5 10 15 Asp Asn Arg MetSer Ala Met Ile Pro Glu Ile Asp 20 25 33 2814 DNA Arabidopsis thaliana33 atgaatcgta tgacgtcaag ctttcttctc ctgacgttcg ccatatgtaa actgatcata 60gccgtgggtc taaacgtggg ccccagtgag ctcctccgca tcggagccat agatgtcgac 120ggccacttca ccgtccaccc ttccgactta gcctccgtct cctcagactt cggtatgctg 180aagtcacctg aagagccatt ggccgtgctt catccatcat cggccgaaga cgtggcacga 240ctcgtcagaa cagcttacgg ttcagccacg gcgtttccgg tctcagcccg aggccacggc 300cattccataa acggacaagc cgcggcgggg aggaacggtg tggtggttga aatgaaccac 360ggcgtaaccg ggacgcccaa gccactcgtc cgaccggatg aaatgtatgt ggatgtatgg 420ggtggagagt tatgggtcga tgtgttgaag aaaacgttgg agcatggctt agcaccaaaa 480tcatggacgg attacttgta tctaaccgtt ggaggtacac tctccaatgc aggaatcagt 540ggtcaagctt ttcaccatgg tcctcaaatt agtaacgtcc ttgagctcga cgttgtaact 600ggttagtatt aaaacattca agttcatata ttttaaatgc ttttgtctga agttttacta 660ataacaagaa attgatacca aaaagtaggg aaaggagagg tgatgagatg ctcagaagaa 720gagaacacaa ggctattcca tggagttctt ggtggattag gtcaatttgg gatcatcact 780cgagcacgaa tctctctcga accagctccc caaagggtaa tattttttta atgactagct 840atcaaaaatc cctggcgggt ccatacgttg taatcttttt agtttttact gttgatggta 900ttttttatat attttggata ataaaaccct aaaatggtat attgtgatga caggtgagat 960ggatacgggt attgtattcg agcttcaaag tgtttacgga ggaccaagag tacttaatct 1020caatgcatgg tcaattaaag tttgattacg tggaaggttt tgtgattgtg gacgaaggac 1080tcgtcaacaa ttggagatct tctttcttct ctccacgtaa ccccgtcaag atctcctctg 1140ttagttccaa cggctctgtt ttgtattgcc ttgagatcac caagaactac cacgactccg 1200actccgaaat cgttgatcag gtcactttca ttattcactt agaaaaaagc gatattttca 1260ttttttatat tgatgaatat ctggaaggat ttaacgctat gcgactattg ggaaatcatt 1320atgaaaaaat atttagttta tatgattgaa agtggtctcc atagtatttt tgttgtgtcg 1380actttattat aacttaaatt tggaagagga catgaagaag aagccagaga ggatctacag 1440agatctagct tttccacctg aacttaataa tgcacattta tataattatt tttcttcttc 1500taaagtttag tttatcacta gcgaattaat catggttact aattaagtag tggacagggt 1560catggaccac tcactcacca aataatgatt cctctttact cttaagttta attttaataa 1620aaccaactct actggaatct taacttatcc ttggttttgg taggctttta tagcaacacg 1680gtttttttaa ttttcctatt ccagattttg tatattaaat gtcgattttt tttctttttg 1740tttcaggaag ttgagattct gatgaagaaa ttgaatttca taccgacatc ggtctttaca 1800acggatttac aatatgtgga ctttctcgac cgggtacaca aggccgaatt gaagctccgg 1860tccaagaatt tatgggaggt tccacaccca tggctcaacc tcttcgtgcc aaaatcaaga 1920atctctgact tcgataaagg cgttttcaag ggcattttgg gaaataaaac aagtggccct 1980attcttatct accccatgaa caaagacaag taagtcttga cattaccatt gattactact 2040tctaaatttc ttctctagaa aaaagaataa aacgagtttt gcattgcatg catgcaaagt 2100tacacttgtg gggattaatt agtggtccaa gaaaaaaagt ttgtcaaaat tgaaaaaaac 2160tagacacgtg gtacatggga ttgtccgaaa aacgttgtcc acatgtgcat cgaaccagct 2220aagattgaca acaacacttc gtcggctcgt atttctcttt ttgttttgtg accaaatccg 2280atggtccaga ttgggtttat ttgtttttaa gttcctagaa ctcatggtgg gtgggtccca 2340atcagattct cctagaccaa accgatctca acgaaccctc cgcacatcat tgattattac 2400attaatatag atattgtcgt tgctgacgtg tcgtaatttg atgttattgt cagatgggac 2460gagaggagct cagccgtgac gccggatgag gaagttttct atctggtggc tctattgaga 2520tcagctttaa cggacggtga agagacacag aagctagagt atctgaaaga tcagaaccgt 2580cggatcttgg agttctgtga acaagccaag atcaatgtga agcagtatct tcctcaccac 2640gcaacacagg aagagtgggt ggctcatttt ggggacaagt gggatcggtt cagaagctta 2700aaggctgagt ttgatccgcg acacatactc gctactggtc agagaatctt tcaaaaccca 2760tctttgtctt tgtttcctcc gtcgtcgtct tcttcgtcag cggcttcatg gtga 2814 34 1620DNA Arabidopsis thaliana 34 atgaatcgta tgacgtcaag ctttcttctc ctgacgttcgccatatgtaa actgatcata 60 gccgtgggtc taaacgtggg ccccagtgag ctcctccgcatcggagccat agatgtcgac 120 ggccacttca ccgtccaccc ttccgactta gcctccgtctcctcagactt cggtatgctg 180 aagtcacctg aagagccatt ggccgtgctt catccatcatcggccgaaga cgtggcacga 240 ctcgtcagaa cagcttacgg ttcagccacg gcgtttccggtctcagcccg aggccacggc 300 cattccataa acggacaagc cgcggcgggg aggaacggtgtggtggttga aatgaaccac 360 ggcgtaaccg ggacgcccaa gccactcgtc cgaccggatgaaatgtatgt ggatgtatgg 420 ggtggagagt tatgggtcga tgtgttgaag aaaacgttggagcatggctt agcaccaaaa 480 tcatggacgg attacttgta tctaaccgtt ggaggtacactctccaatgc aggaatcagt 540 ggtcaagctt ttcaccatgg tcctcaaatt agtaacgtccttgagctcga cgttgtaact 600 gggaaaggag aggtgatgag atgctcagaa gaagagaacacaaggctatt ccatggagtt 660 cttggtggat taggtcaatt tgggatcatc actcgagcacgaatctctct cgaaccagct 720 ccccaaaggg tgagatggat acgggtattg tattcgagcttcaaagtgtt tacggaggac 780 caagagtact taatctcaat gcatggtcaa ttaaagtttgattacgtgga aggttttgtg 840 attgtggacg aaggactcgt caacaattgg agatcttctttcttctctcc acgtaacccc 900 gtcaagatct cctctgttag ttccaacggc tctgttttgtattgccttga gatcaccaag 960 aactaccacg actccgactc cgaaatcgtt gatcaggaagttgagattct gatgaagaaa 1020 ttgaatttca taccgacatc ggtctttaca acggatttacaatatgtgga ctttctcgac 1080 cgggtacaca aggccgaatt gaagctccgg tccaagaatttatgggaggt tccacaccca 1140 tggctcaacc tcttcgtgcc aaaatcaaga atctctgacttcgataaagg cgttttcaag 1200 ggcattttgg gaaataaaac aagtggccct attcttatctaccccatgaa caaagacaaa 1260 tgggacgaga ggagctcagc cgtgacgccg gatgaggaagttttctatct ggtggctcta 1320 ttgagatcag ctttaacgga cggtgaagag acacagaagctagagtatct gaaagatcag 1380 aaccgtcgga tcttggagtt ctgtgaacaa gccaagatcaatgtgaagca gtatcttcct 1440 caccacgcaa cacaggaaga gtgggtggct cattttggggacaagtggga tcggttcaga 1500 agcttaaagg ctgagtttga tccgcgacac atactcgctactggtcagag aatctttcaa 1560 aacccatctt tgtctttgtt tcctccgtcg tcgtcttcttcgtcagcggc ttcatggtga 1620 35 539 PRT Arabidopsis thaliana 35 Met AsnArg Met Thr Ser Ser Phe Leu Leu Leu Thr Phe Ala Ile Cys 1 5 10 15 LysLeu Ile Ile Ala Val Gly Leu Asn Val Gly Pro Ser Glu Leu Leu 20 25 30 ArgIle Gly Ala Ile Asp Val Asp Gly His Phe Thr Val His Pro Ser 35 40 45 AspLeu Ala Ser Val Ser Ser Asp Phe Gly Met Leu Lys Ser Pro Glu 50 55 60 GluPro Leu Ala Val Leu His Pro Ser Ser Ala Glu Asp Val Ala Arg 65 70 75 80Leu Val Arg Thr Ala Tyr Gly Ser Ala Thr Ala Phe Pro Val Ser Ala 85 90 95Arg Gly His Gly His Ser Ile Asn Gly Gln Ala Ala Ala Gly Arg Asn 100 105110 Gly Val Val Val Glu Met Asn His Gly Val Thr Gly Thr Pro Lys Pro 115120 125 Leu Val Arg Pro Asp Glu Met Tyr Val Asp Val Trp Gly Gly Glu Leu130 135 140 Trp Val Asp Val Leu Lys Lys Thr Leu Glu His Gly Leu Ala ProLys 145 150 155 160 Ser Trp Thr Asp Tyr Leu Tyr Leu Thr Val Gly Gly ThrLeu Ser Asn 165 170 175 Ala Gly Ile Ser Gly Gln Ala Phe His His Gly ProGln Ile Ser Asn 180 185 190 Val Leu Glu Leu Asp Val Val Thr Gly Lys GlyGlu Val Met Arg Cys 195 200 205 Ser Glu Glu Glu Asn Thr Arg Leu Phe HisGly Val Leu Gly Gly Leu 210 215 220 Gly Gln Phe Gly Ile Ile Thr Arg AlaArg Ile Ser Leu Glu Pro Ala 225 230 235 240 Pro Gln Arg Val Arg Trp IleArg Val Leu Tyr Ser Ser Phe Lys Val 245 250 255 Phe Thr Glu Asp Gln GluTyr Leu Ile Ser Met His Gly Gln Leu Lys 260 265 270 Phe Asp Tyr Val GluGly Phe Val Ile Val Asp Glu Gly Leu Val Asn 275 280 285 Asn Trp Arg SerSer Phe Phe Ser Pro Arg Asn Pro Val Lys Ile Ser 290 295 300 Ser Val SerSer Asn Gly Ser Val Leu Tyr Cys Leu Glu Ile Thr Lys 305 310 315 320 AsnTyr His Asp Ser Asp Ser Glu Ile Val Asp Gln Glu Val Glu Ile 325 330 335Leu Met Lys Lys Leu Asn Phe Ile Pro Thr Ser Val Phe Thr Thr Asp 340 345350 Leu Gln Tyr Val Asp Phe Leu Asp Arg Val His Lys Ala Glu Leu Lys 355360 365 Leu Arg Ser Lys Asn Leu Trp Glu Val Pro His Pro Trp Leu Asn Leu370 375 380 Phe Val Pro Lys Ser Arg Ile Ser Asp Phe Asp Lys Gly Val PheLys 385 390 395 400 Gly Ile Leu Gly Asn Lys Thr Ser Gly Pro Ile Leu IleTyr Pro Met 405 410 415 Asn Lys Asp Lys Trp Asp Glu Arg Ser Ser Ala ValThr Pro Asp Glu 420 425 430 Glu Val Phe Tyr Leu Val Ala Leu Leu Arg SerAla Leu Thr Asp Gly 435 440 445 Glu Glu Thr Gln Lys Leu Glu Tyr Leu LysAsp Gln Asn Arg Arg Ile 450 455 460 Leu Glu Phe Cys Glu Gln Ala Lys IleAsn Val Lys Gln Tyr Leu Pro 465 470 475 480 His His Ala Thr Gln Glu GluTrp Val Ala His Phe Gly Asp Lys Trp 485 490 495 Asp Arg Phe Arg Ser LeuLys Ala Glu Phe Asp Pro Arg His Ile Leu 500 505 510 Ala Thr Gly Gln ArgIle Phe Gln Asn Pro Ser Leu Ser Leu Phe Pro 515 520 525 Pro Ser Ser SerSer Ser Ser Ala Ala Ser Trp 530 535 36 842 DNA Arabidopsis thaliana 36aagcttaaat gacaatttag taccttgggt tggtcatgat ttagagcgga acaaatatac 60catacatcaa acgaggatat acagagaaaa ttcatggaag tatggaattt agaggacaat 120ttctcttctg ggctacaacg gaccggccca ttcgctcatt tacccagagg tatcgagttt 180gtggactttt gatgccgcta gagactattg gcatcggatt gaaaaaaatg tttacttcgt 240tgttaacaat tttctgaatg caatattttc cttgtcatga atatttaaac ttgttattac 300tttcttttag cttaggtgtg gacaattatg gagtttactt caaacgagga agaatcttaa 360acgctcggtt caggtctcga aaacaaacca actcacaatc ctgacttaat tgaggaaaac 420aatgcaaaac cacatgcatg cttccatatt tctatcataa tcttataaga aaaaacacta 480ctaagtgaaa tgattctgta tatatataac caatgccttt tgttttgtga tattttatgt 540atatataact attgactttt gtcatctatg gatagtgtct cgggctcttg gcaaacatat 600ttcaaagaaa agttaatgac tgtaattaat taatctgaag ctagaaacag aaccccgagg 660taaaagaaaa agacagagca catgaagttt agtactttta tatatttaat atatcattct 720ttcttattgc ttatctctaa agcaaaaact tccctaaacc ctaagccaaa ggactcagat 780cgatgcagaa ccaagaaggc ttgttttgga tttgagagcc aaatgcaaag aaaaaaactc 840 tt842

1. Use of a nucleic add encoding a plant cytokinin oxidase or encoding aprotein that reduces the level of active cytokinins in plants or plantparts for stimulating root growth or for enhancing the formation oflateral or adventitious roots or for altering root geotropism.
 2. Amethod for stimulating root growth or for enhancing the formation oflateral or adventitious roots or for altering root geotropism comprisingexpression of a nucleic acid encoding a plant cytokinin oxidase selectedfrom the group consisting of: (a) nucleic acids comprising a DNAsequence as given in any of SEQ ID NOs 27, 1, 3, 5, 7, 9, 11, 25, 26, 28to 31, 33 or 34, or the complement thereof, (b) nucleic acids comprisingthe RNA sequences corresponding to any of SEQ ID NOs 27, 1, 3, 5, 7, 9,11, 25, 26, 28 to 31, 33 or 34, or the complement thereof, (c) nucleicacids specifically hybridizing to any of SEQ ID NOs 27, 1, 3, 5, 7, 9,11, 25, 26, 28 to 31, 33 or 34, or to the complement thereof, (d)nucleic acids encoding a protein comprising the amino acid sequence asgiven in any of SEQ ID NOs 2, 4, 6, 8, 10, 12, 32 or 35, or thecomplement thereof, (e) nucleic acids as defined in any of (a) to (d)characterized in that said nucleic acid is DNA, genomic DNA, cDNA,synthetic DNA or RNA wherein T is replaced by U, (f) a nucleic acidwhich is degenerated to a nucleic acid as given in any of SEQ ID NOs 27,1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33 or 34, or which is degeneratedto a nucleic acid as defined in any of (a) to (e) as a result of thegenetic code, (g) nucleic acids which are diverging from a nucleic acidencoding a protein as given in any of SEQ ID NOs 2, 4, 6, 8, 10, 12 or35 or which is diverging from a nucleic acid as defined in any of (a) to(e), due to the differences in codon usage between the organisms, (h)nucleic acids encoding a protein as given in SEQ ID NOs 2, 4, 6, 8, 10,12 or 35 or nucleic acids as defined in (a) to (e) which are divergingdue to the differences between alleles, (i) nucleic acids encoding aprotein as given in any of SEQ ID NOs 2, 4, 6, 8, 10, 12 or 35, (j)functional fragments of nucleic acids as defined in any of (a) to (i)having the biological activity of a cytokinin oxidase, and (k) nucleicacids encoding a plant cytokinin oxidase, or comprising expression,preferably in roots, of a nucleic acid encoding a protein that reducesthe level of active cytokinins in plants or plant parts.
 3. An isolatednuclei acid encoding a novel plant protein having cytokinin oxidaseactivity selected from the group consisting of: (a) a nucleic acidcomprising a DNA sequence as given in any of SEQ ID NOs 29, 3, 5, 9, 26,27, 31, 33 or 34, or the complement thereof, (b) a nucleic acidcomprising the RNA sequences corresponding to any of SEQ ID NOs 29, 3,5, 9, 26, 27, 31, 33 or 34, or the complement thereof, (c) a nucleicacid specifically hybridizing to a nucleic acid as given in any of SEQID NOs 29, 3, 5, 9, 26, 27, 31, 33 or 34, or the complement thereof, (d)a nucleic acid encoding a protein with an amino acid sequence comprisingthe polypeptide as given in SEQ ID NO 32 and which is at least 70%similar to the amino acid sequence as given in SEQ ID NO 4, (e) anucleic acid encoding a protein with an amino acid sequence which is atleast 47% similar to the amino acid sequence as given in SEQ ID NO 6,(f) a nucleic acid encoding a protein with an amino acid sequence whichis at least 47% similar to the amino acid sequence as given in SEQ ID NO10 or 35, (g) a nucleic acid encoding a protein comprising the aminoacid sequence as given in any of SEQ ID NOs 4, 6, 10, 32 or 35, (h) anucleic acid which is degenerated to a nucleic acid as given in any ofSEQ ID NOs 29, 3, 5, 9, 26, 27, 33 or 34 or which is degenerated to anucleic acid as defined in any of (a) to (g) as a result of the geneticcode, (i) a nucleic acid which is diverging from a nucleic acid encodinga protein as given in any of SEQ ID NOs 4, 6, 10 or 35 or which isdiverging from a nucleic acid as defined in any of (a) to (g) due to thedifferences in codon usage between the organisms, (j) a nucleic acidencoding a protein as given in SEQ ID NOs 4, 6, 10 or 35, or a nucleicacid as defined in (a) to (g) which is diverging due to the differencesbetween alleles, (k) a nucleic acid encoding an immunologically activefragment of a cytokinin oxidase encoded by a nucleic acid as given inany of SEQ ID NOs 29, 3, 5, 9, 26, 27, 31, 33 or 34, or animmunologically active fragment of a nucleic acid as defined in any of(a) to (j), (l) a nucleic acid encoding a functional fragment of acytokinin oxidase encoded by a nucleic acid as given in any of SEQ IDNOs 29, 3, 5, 9, 26, 27, 31, 33 or 34, or a functional fragment of anucleic acid as defined in any of (a) to (j), wherein said fragment hasthe biological activity of a cytokinin oxidase, and (m) a nucleic acidencoding a protein as defined in SEQ ID NO 4, 6, 10 or 35, provided thatsaid nucleic acid is not the nucleic acid as deposited under any of thefollowing Genbank accession numbers: AC005917, AB024035, and AC023754.4. An isolated nucleic acid according to claim 3 which is DNA, cDNA,genomic DNA or synthetic DNA, or RNA wherein T is replaced by U.
 5. Anucleic acid molecule of at least 15 nucleotides in length hybridizingspecifically with a nucleic acid of claim 3 or
 4. 6. A nucleic acidmolecule of at least 15 nucleotides in length specifically amplifying anucleic acid of claim 3 or
 4. 7. A vector comprising a nucleic acid ofclaim 3 or
 4. 8. A vector according to claim 7 which is an expressionvector wherein the nucleic acid is operably linked to one or morecontrol sequences allowing the expression of said nucleic acid inprokaryotic and/or eukaryotic host cells.
 9. A host cell containing anucleic acid according to claim 3 or 4 or a vector according to claim 7or
 8. 10. The host cell of claim 9, wherein the host cell is abacterial, insect, fungal, plant or animal cell.
 11. An isolatedpolypeptide encodable by a nucleic acid of claim 3 or 4, or a homologueor a derivative thereof, or an immunologically active or a functionalfragment thereof.
 12. The polypeptide of claim 11 which has an aminoacid sequence as given in any of SEQ ID NOs 4, 6, 10 or 35, or ahomologue or a derivative thereof, or an immunologically active or afunctional fragment thereof.
 13. A method for producing a polypeptideaccording to claim 11 or 12 comprising culturing a host cell of claim 9or 10 under conditions allowing the expression of the polypeptide andrecovering the produced polypeptide from the culture.
 14. An antibodyspecifically recognizing a polypeptide of claim 11 or 12 or a specificepitope thereof.
 15. A method for the production of transgenic plants,plant cells or plant tissues comprising the introduction of a nucleicacid of claim 3 or 4 in an expressible format or a vector of claim 7 or8 in said plant, plant cell or plant tissue.
 16. A method for theproduction of altered plants, plant cells or plant tissues comprisingthe introduction of a polypeptide of claim 11 or 12 directly into acell, a tissue or an organ of said plant.
 17. A method for effecting theexpression of a polypeptide of claim 11 or 12 comprising theintroduction of a nucleic acid of claim 3 or 4 operably linked to one ormore control sequences or a vector of claim 7 or 8 stably into thegenome of a plant cell.
 18. The method of claim 16 or 17 furthercomprising regenerating a plant from said plant cell.
 19. A transgenicplant cell comprising a nucleic acid of claim 3 or 4 which is operablylinked to regulatory elements allowing transcription and/or expressionof said nucleic acid in plant cells or a transgenic plant cellobtainable by a method of claim 16 or
 17. 20. The transgenic plant cellof claim 18 wherein said nucleic acid of claim 3 or 4 is stablyintegrated into the genome of said plant cell.
 21. A transgenic plant orplant tissue comprising plant cells of claim 19 or
 20. 22. A harvestablepart of a plant of claim
 21. 23. The harvestable part of a plant ofclaim 22 which is selected from the group consisting of seeds, leaves,fruits, stem cultures, rhizomes, roots, tubers and bulbs.
 24. Theprogeny derived from any of the plants or plant parts of any of claims21 to
 23. 25. A method for stimulating root growth comprising expressionof a nucleic acid of claim 3 or 4 or a nucleic acid as defined in claim2 or comprising expression of another protein that reduces the level ofactive cytokinins in plants or plant parts.
 26. A method for enhancingthe formation of lateral or adventitious roots comprising expression ofa nucleic acid of claim 3 or 4 or a nucleic acid as defined in claim 2or comprising expression of another protein that reduces the level ofactive cytokinins in plants or plant parts.
 27. A method for alteringroot geotropism comprising altering the expression of a nucleic acid ofclaim 3 or 4 or a nucleic acid as defined in claim 2 or comprisingexpression of another protein that reduces the level of activecytokinins in plants or plant parts.
 28. A method of any of claims 25 to27, said method leading to an increase in yield.
 29. The method of anyof claims 25 to 28 wherein said expression of said nucleic acid occursunder the control of a strong constitutive promoter.
 30. The method ofany of claims 25 to 28 wherein said expression of said nucleic acidoccurs under the control of a promoter that is preferentially expressedin roots.
 31. A method for identifying and obtaining proteinsinteracting with a polypeptide of claim 11 or 12 comprising a screeningassay wherein a polypeptide of claim 11 or 12 is used.
 32. The method ofclaim 31 comprising a two-hybrid screening assay wherein a polypeptideof claim 11 or 12 as a bait and a cDNA library as prey are used.
 33. Amethod for modulating the interaction between a polypeptide of claim 11or 12 and interacting protein partners obtainable by a method accordingto claim 31 or
 32. 34. A method for Identifying and obtaining compoundsinteracting with a polypeptide of claim 11 or 12 comprising the stepsof: a) providing a two-hybrid system wherein a polypeptide of claim 11or 12 and an interacting protein partner obtainable by a methodaccording to claim 31 or 32 are expressed, b) interacting said compoundwith the complex formed by the expressed polypeptides as defined in (a),and, c) performing measurement of interaction of said compound with saidpolypeptide or the complex formed by the expressed polypeptides asdefined in (a).
 35. A method for identifying compounds or mixtures ofcompounds which specifically bind to a polypeptide of claim 11 or 12,comprising: a) combining a polypeptide of claim 11 or 12 with saidcompound or mixtures of compounds under conditions suitable to allowcomplex formation, and, b) detecting complex formation, wherein thepresence of a complex identifies a compound or mixture whichspecifically binds said polypeptide.
 36. A method of any of claims 31 to35 wherein said compound or mixture Inhibits the activity of saidpolypeptide of claim 11 or 12 and can be used for the rational design ofchemicals.
 37. Use of a compound or mixture identified by means of amethod of any of claims 31 to 35 as a plant growth regulator orherbicide.
 38. A method for production of a plant growth regulator orherbicide composition comprising the steps of the method of any ofclaims 31 to 35 and formulating the compounds obtained from said stepsin a suitable form for the application in agriculture or plant cell ortissue culture.
 39. A method for the design of or screening forgrowth-promoting chemicals or herbicides comprising the use of a nucleicacid of claim 3 or 4 or a nucleic acid as defined in claim 2 or a vectorof claim 7 or
 8. 40. Use of a nucleic acid molecule of claim 3 or 4 or anucleic acid as defined in claim 2, the vector of claim 7 or 8, apolypeptide of claim 11 or 12 for increasing yield.
 41. Use of a nucleicacid molecule of claim 3 or 4 or a nucleic acid as defined in claim 2,the vector of claim 7 or 8, a polypeptide of claim 11 or 12 forstimulating root growth.
 42. Use of a nucleic acid molecule of claimclaim 3 or 4 or a nucleic acid as defined in claim 2, the vector ofclaim 7 or 8, a polypeptide of claim 11 or 12 for enhancing theformation of lateral or adventitious roots.
 43. Use of a nucleic acidmolecule of claim claim 3 or 4 or a nucleic acid as defined in claim 2,the vector of claim 7 or 8, a polypeptide of claim 11 or 12 for alteringroot geotropism.
 44. Diagnostic composition comprising at least anucleic acid molecule of any of claims 3 to 6, the vector of claim 7 or8, a polypeptide of claim 11 or 12 or an antibody of claim
 14. 45. Amethod for increasing the size of the root meristem comprisingexpression of a nucleic acid of claim 3 or 4 or a nucleic acid asdefined in claim 2 or comprising expression of a nucleic acid encoding aprotein that reduces the level of active cytokinins in plants or plantparts, preferably in roots.
 46. A method for increasing the root sizecomprising expression of a nucleic acid of claim 3 or 4 or a nucleicacid as defined in claim 2 or comprising expression of another nucleicacid encoding a protein that reduces the level of active cytokinins inplants or plant parts, preferably in roots.
 47. A method for increasingthe size of the shoot meristem comprising downregulation of expressionof a nucleic acid of claim 3 or 4 or a nucleic acid as defined in claim2, preferably in shoots.
 48. A method for delaying leaf senescencecomprising downregulation of expression of a nucleic acid of claim 3 or4 or a nucleic acid as defined in claim 2 in leaves, preferably insenescing leaves.
 49. A method for altering leaf senescence comprisingexpression of a nucleic acid of claim 3 or 4 or a nucleic acid asdefined in claim 2 in senescing leaves.
 50. A method for increasing leafthickness comprising expression of a nucleic acid of claim 3 or 4 or anucleic acid as defined in claim 2 or comprising expression of a nucleicacid encoding a protein that reduces the level of active cytokinins inplants or plant parts.
 51. A method for reducing the vessel sizecomprising expression of a nucleic acid of claim 3 or 4 or a nucleicacid as defined in claim 2 or comprising expression of a nucleic acidencoding a protein that reduces the level of active cytokinins in plantsor plant parts.
 52. A method for increasing the vessel size comprisingdownregulation of expression of a nucleic acid of claim 3 or 4 or anucleic acid as defined in claim 2 in plants or plant parts.
 53. Amethod for inducing parthenocarpy comprising expression of a nucleicacid of claim 3 or 4 or a nucleic acid as defined in claim 2 orcomprising expression of a nucleic acid encoding a protein that reducesthe level of active cytokinins in plants or plant parts, preferably inthe placenta, ovules and tissues derived therefrom.
 54. A method forimproving standability of seedlings comprising expression of a nucleicacid of claim 3 or 4 or a nucleic acid as defined in claim 2 orcomprising expression of a nucleic acid encoding a protein that reducesthe level of active cytokinins in seedlings, preferably in the roots ofseedlings.
 55. A method for increasing branching comprising expressionof a nucleic acid of claim 3 or 4 or a nucleic acid as defined in claim2 in plants or plant parts.
 56. A method for improving lodgingresistance comprising expression of a nucleic acid of claim 3 or 4 or anucleic acid as defined in claim 2 in plants or plant parts, preferablyin stems or axillary buds.
 57. Use of a transgenic rootstock in graftingprocedures with a scion for improving the root-related characteristicsof the resulting plant or tree characterized in an enhanced root growthdue to expression of a plant cytokinin oxidase in said rootstock. 58.Use according to claim 57 wherein said plant cytokinin oxidase isencoded by a nucleic acid of claim 3 or 4 or a nucleic acid as definedin claim
 2. 59. A transgenic plant comprising a transgenic rootstockexpressing a plant cytokinin oxidase according to claim 57 or 58 andfurther comprising a scion.
 60. A harvestable part of a plant of claim59.
 61. A method for stimulating root growth and development comprisingexpression of a nucleic acid encoding a plant cytokinin oxidase in atransgenic plant cell or tissue culture.
 62. A method according to claim61 wherein said nucleic acid is at least one of the nucleic acids ofclaim 3 or as defined in claim 2.